Tissue protective peptides and uses thereof

ABSTRACT

Provided herein are tissue protective peptides derived from or sharing consensus sequences with portions of cytokine receptor ligands, including Erythropoietin (EPO), that are generally located on or within the region of the cytokine receptor ligand that faces away from a receptor complex while the ligand is bound to the receptor. Also provide herein are fragments, chimeras, as well as peptides designed to mimic the spatial localization of key amino acid residues within the tissue protective receptor ligands, e.g., EPO; methods for treating or preventing a disease or disorder using tissue protective peptides; and methods for enhancing excitable tissue function using tissue protective peptides.

1. INTRODUCTION

The present invention is directed to novel tissue protective peptides.The tissue protective peptides of the invention may bind to a tissueprotective receptor complex. In particular, the present invention isdrawn to tissue protective peptides derived from or sharing consensussequences with portions of cytokine receptor ligands, includingErythropoietin (EPO), that are not involved in the binding of the ligandto the receptor complex, e.g., to the EPO receptor homodimer.Accordingly, the tissue protective peptides of the invention are derivedfrom the amino acid sequences of regions of cytokine receptor ligandsthat are generally located on or within the region of the ligand proteinthat is opposite of the receptor complex, i.e., are generally derivedfrom amino acid sequences of regions of the ligand protein that faceaway from the receptor complex while the ligand is bound to thereceptor. The invention is further directed to the consensus sequencesfor use in engineering a synthetic tissue protective peptide. Thesetissue protective peptides also include fragments, chimeras, as well aspeptides designed to mimic the spatial localization of key amino acidresidues within the tissue protective receptor ligands, e.g., EPO.

The invention also encompasses methods for treating, preventing orameliorating a disease or disorder and or treating, restoring orameliorating a tissue injury using tissue protective peptides of thecurrent invention. The invention also encompasses methods for enhancingexcitable tissue function using tissue protective peptides of thecurrent invention.

2. BACKGROUND OF THE INVENTION

Erythropoietin (“EPO”) is a glycoprotein hormone commonly associatedwith the maintenance of hematocrit and, more recently, tissueprotection. Mature human EPO protein comprises 165 amino acids and has amolecular weight of 34 kDa, with glycosyl residues contributing about40% of the weight of the molecule. The EPO molecule comprises fourhelices that interact via their hydrophobic domains to form apredominantly globular structure within an aqueous environment (Cheethamet al., 1998, Nat. Struct. Biol. 5:861-866, which is hereby incorporatedby reference in its entirety). The invention derives from the discoverythat certain amino acids facing the aqueous environment (i.e., away fromthe hydrophobic, globular central core) mediate tissue protection.Peptides can be derived or designed from an understanding of the tissueprotective regions that have been identified by the Applicants.

As noted above, EPO is pluripotent. In its hormonal role, EPO regulateshematocrit through its role in the maturation of erythroid progenitorcells into erythrocytes. EPO acts as an anti-apoptotic agent during thematuration process of erythroid progenitor cells, permitting progenitorcells to mature into erythrocytes. Decreased levels of tissue oxygen(hypoxia) trigger an increased production of erythropoietin by thekidney, which results in increased erythropoiesis. Given that the kidneynormally produces the majority of the serum erythropoietin, the loss ofkidney function, such as in chronic renal failure, results in decreasedproduction of EPO and often anemia. Similarly, anemia may result fromother chronic conditions, such as cancer, or treatments associated withthese illnesses, such as chemotherapy, which directly suppress theproduction of EPO. Commercially available recombinant erythropoietin hasbeen available under the trademarks of PROCRIT, available from OrthoBiotech Inc., Raritan, N.J., and EPOGEN, available from Amgen, Inc.,Thousand Oaks, Calif. and has been used to treat anemia resulting fromend stage renal disease, therapy with AZT (zidovudine) in HIV-infectedpatients, oncology patients, and chemotherapy. Currently ahyperglycosylated erythropoietin, ARANESP™ (Amgen, Thousand Oaks,Calif.), is available for the treatment of anemia. Additionally, thesecompounds have been used to increase the hematocrits of patientsundergoing surgery to reduce the need for allogenic blood transfusions.

Recently, several lines of evidence have suggested that EPO alsofunctions locally in a paracrine-autocrine manner to minimize tissuedamage. For example, EPO improves an hypoxic cellular microenvironmentand decreases programmed cell death caused by metabolic stress. Both ofthese activities are moderated, in part, through EPO's interaction witha specific cell surface receptor comprised, in part, by theerythropoietin receptor (“EPOR”) protein. EPOR is an approximately 66kDa protein and is a member of the Type-1 cytokine receptor family. Thisfamily comprises receptors that are grouped together based on the sharedhomology of their extracellular domains and includes receptors forinterleukin IL-2, IL3, IL4, IL5, IL6, IL7, IL9, IL11, granulocytemacrophage—colony stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), leukemia inhibiting factor (LIF), ciliaryneurotrophic factor (CNTF), thrombopoietin, growth hormone andprolactin. The conserved extracellular domain of these receptors has alength of approximately 200 amino acids, comprises four positionallyconserved cysteine residues in the amino-terminal region (Cys 294, Cys283, Cys 248, and Cys 238, which appear to be critical to themaintenance and the structural integrity of the receptors (Murray, 1996,Harpers Biochemistry 24^(th) ed. pp. 524-526, Appilion & Lange, Ltd.;Caravella et al., 1996, Protein: Struct. Funct. Gen. 24:394-401, each ofwhich is hereby incorporated by reference in its entirety)), and aTrp-Ser-X-Trp-Ser (SEQ ID NO:58) motif located proximal to thetransmembrane domain.

In connection with erythropoiesis, EPOR functions in a manner similar toother receptors within the Type-1 cytokine receptor family. First, thereceptor ligand, e.g., EPO, binds to a preformed dimer of EPOR, (EPOR)₂.It has been determined that EPO interacts with the extracellular domainof the classic (EPOR)₂ homodimer receptor via two distinct regions onthe ligand surface: a high affinity receptor binding site (site)) and alow affinity receptor binding site (site 2). The amino acid sequences ofEPO associated with site 1 are TKVNFY, SEQ ID NO:2, corresponding toamino acids 44-49 of SEQ ID NO:1, and SNFLRG, SEQ ID NO:3, correspondingto amino acids 146-151 of SEQ ID NO:1; the sequences associated withsite 2 are VLERY, SEQ ID NO:4, corresponding to amino acids 11-15 of SEQID NO:1, and SGLRS, SEQ NO:5, corresponding to amino acids 100-104 ofSEQ ID NO:1 (Cheetham et al., 1998, Nature Structural Biology 5:861-866,hereby incorporated by reference in its entirety). EPOR homodimeractivation leads to tyrosine phosphorylation of signaling proteins thatare associated with EPOR, e.g., Jak2 tyrosine kinases, that may in turnactivate several different pathways including, for example, thephosphatidylinositol (PI) 3-kinase pathway, the Ras/MAP kinase pathway,and/or the STAT pathway. These pathways trigger the anti-apoptoticfunctions necessary for erythropoiesis that are mediated byerythropoietin (Kirito et al., 2002, Blood 99:102-110; Livnah et al.,1999, Science 283:987-990; Naranda et al., 2002, Endocrinology143:2293-2302; Remy et al., 1999, Science 283:990-993; and Yoshimura etal., 1996, The Oncologist 1:337-339, each of which is herebyincorporated by reference in its entirety).

Recently, Applicants have discovered that the tissue protectiveproperties of EPO are mediated by a receptor that comprises not onlyEPOR but also another receptor protein, the beta common receptor(“β_(c)”). The EPOR/β_(c) receptor is, in contrast to the homodimer(EPOR)₂, a heterocomplex (see infra) and is known to play a role in theprotection of excitable tissues. See, e.g., WO 2004/096148 and PCT no.PCT/US01/49479, filed Dec. 28, 2001, U.S. patent application Ser. No.09/753,132, filed Dec. 29, 2000, and 10/188,905, filed Jul. 3, 2002,each of which is hereby incorporated by reference in its entirety.Although Applicants had established that the β_(c) receptor is centralto the tissue protective pathways in these excitable tissues, thestructure of the activating ligands for the receptors was still unknown.

3. SUMMARY

The present invention is drawn to isolated polypeptides that have atleast one cellular protective activity in a responsive cell, tissue, ororgan, which polypeptides contain amino acid motifs comprising theconsensus sequence (a) H₁—N₁—(X)_(n)—N₂—H₂, wherein _(n) is 0, 1, 2, 3,4 or 5; (b) H₁—N₁—(X)_(n)—N₂-L₁, wherein _(n) is 0, 1, 2, 3, 4 or 5; (c)L₁-N₁—(X)_(n)—N₂—H₁, wherein _(n) is 0, 1, 2, 3, 4 or 5; (d)H₁—N₁-(L)_(n)-P₁—H₂, wherein _(n) is 0 or 1; or (e) H₁—P₁-(L)_(n)-N₁—H₂,wherein _(n) is 0 or 1, and wherein H₁ and H₂ are hydrophobic aminoacids, N₁ and N₂ are negatively charged amino acids, X is any aminoacid, L₁ is a polar amino acid, and P₁ is a positively charged aminoacid. In certain embodiments, the peptides of the invention also lackerythoropoietic activity, e.g., do not increase hemoglobin or hematocritin a recipient. In further embodiments, the isolated polypeptides of theinvention consist of no more than 10, no more than 15, no more than 20,or no more than 30 amino acids. In other embodiments, the isolatedpeptide has less than 90%, less than 85%, less than 80%, less than 75%,less than 70%, less than 65%, less than 60%, less than 55%, less than50%, less than 45%, less than 40%, less than 35%, less than 30%, or lessthan 20 percent sequence identity with any portion of the amino acidsequence of mature human erythropoietin (“EPO”) set forth in SEQ ID NO:1, wherein said portion of EPO contains the same number of amino acidresidues as said peptide.

In certain embodiments of the invention described hereinabove, whereinthe isolated polypeptide comprises the structural motif (a)H₁—N₁—(X)_(n)—N₂—H₂, wherein _(n) is 0, 1, 2, 3, 4 or 5 (embodied bysequence identifiers 6-11, respectively, discussed infra); (b)H₁—N₁-(L)_(n)-P₁—H₂, wherein _(n) is 0 or 1 (embodied by sequenceidentifiers 24-25, respectively, discussed infra); or (e)H₁—P_(n)-(L)_(n)-N₁—H₂, wherein _(n) is 0 or 1 (embodied by sequenceidentifiers 26-27, respectively, discussed infra), H₁ and H₂ may be thesame hydrophobic amino acid. In other embodiments of the inventiondescribed hereinabove, wherein the isolated polypeptide comprises thestructural motifs (a) H₁—N₁—(X)_(n)—N₂—H₂, wherein _(n) is 0, 1, 2, 3, 4or 5; (d) H₁—N₁-(L)_(n)-P₁—H₂, wherein _(n) is 0 or 1; or (e)H₁—P₁-(L)_(n)-N₁—H₂, wherein _(n) is 0 or 1, H₁ and H₂ may be differenthydrophobic amino acids. In other embodiments, the invention providesfor an isolated polypeptide comprising the amino acid motif (a)H₁—N₁—(X)_(n)—N₂—H₂, wherein _(n) is 0, 1, 2, 3, 4 or 5; (b)H₁—N₁—(X)_(n)—N₂-L₁, wherein _(n) is 0, 1, 2, 3, 4 or 5; (c)L₁-N₁—(X)_(n)—N₂—H₁, wherein _(n) is 0, 1, 2, 3, 4 or 5, and wherein N₁and N₂ may the same or may be different negatively charged amino acids.

The invention provides for isolated polypeptides comprising the aminoacid motifs described hereinabove, wherein said motifs are formed byconsecutive amino acids within the amino-acid sequence of saidpolypeptide. In specific examples in accordance with this embodiment,the invention provides for an isolated polypeptide comprising the aminoacid motif H₁—N₁—N₂—H₂ (SEQ ID NO:6), H₁—N₁—X—N₂—H₂ (SEQ ID NO:7),H₁—N₁—X—X—N₂—H₂ (SEQ ID NO:8), H₁—N₁—X—X—X—N₂—H₂ (SEQ ID NO:9),H₁—N₁—X—X—X—X—N₂—H₂ (SEQ ID NO:10), H₁—N₁—X—X—X—X—X—N₂—H₂ (SEQ IDNO:11), H₁—N₁—N₂-L₁ (SEQ ID NO:12), H₁—N₁—X—N₂-L₁ (SEQ ID NO:13), (SEQID NO:14), H₁—N₁—X—X—X—N₂—L₁ (SEQ ID NO:15), H₁—N₁—X—X—X—X—N₂—L₁ (SEQ IDNO:16), H₁—N₁—X—X—X—X—X—N₂—L₁ (SEQ ID NO:17), L₁-N₁—N₂—H₂ (SEQ IDNO:18), L₁-N₁—X—N₂—H₂ (SEQ ID NO:19), L₁-N₁—X—X—N₂—H₂ (SEQ ID NO:20),L₁-N₁—X—X—X—N₂—H₂ (SEQ ID NO:21), L₁-N₁—X—X—X—X—N₂—H₂ (SEQ ID NO:22),L₁-N₁—X—X—X—X—X—N₂—H₂ (SEQ ID NO:23), H₁—N₁—P₁—H₂ (SEQ ID NO:24),H₁—N₁-L₁-P₁—H₂ (SEQ ID NO:25), H₁—P₁—N₁—H₂ (SEQ ID NO:26), orH₁—P₁-L₁-N₁—H₂ (SEQ ID NO:27), wherein H₁ and H₂ are hydrophobic aminoacids, N₁ and N₂ are negatively charged amino acids, X is any aminoacid, L₁ is a polar amino acid, and P₁ is a positively charged aminoacid. In certain aspects consistent with this embodiment, wherein theisolated polypeptide comprises a motif having the amino acid residues H₁and H₂, H₁ and H₂ may the same or may be different hydrophobic aminoacids. In other aspects consistent with this embodiment, wherein theisolated polypeptide comprises a motif having the amino acid residues N₁and N₂, N₁ and N₂ may the same or may be different negatively chargedamino acids.

In other embodiments, the invention provides isolated polypeptideswherein the amino acid motif is formed due to the spatial organizationof amino acids within the tertiary structure of a polypeptide, i.e., theamino acids forming the motif are spatially adjacent to one another inthe three dimensional structure, i.e. tertiary structure, of thepolypeptide but may be separated by 1 or more amino acids within theprimary amino acid sequence of the polypeptide chain. In a specificexample in accordance with this embodiment, the amino acid motifcomprising amino acid residues H₁, N₁, N₂, and H₂ analogous to SEQ IDNO:6, discussed supra, may form as a result of the tertiary structureadopted by, i.e., protein folding of peptides comprising, e.g., SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, whereinthe amino acid residues between N₁ and N₂, e.g. (X)_(n), fold such thatN₁ and N₂ become linearly adjacent. Accordingly, the inventionencompasses isolated peptides comprising the amino acid motif H₁N₁N₂H₂;H₁N₁N₂L₁; L₁N₁N₂H₁; H₁N₁(L)_(n)P₁H₂, wherein n is 0 or 1; orH₁P₁(L)_(n)N₁H₂, wherein n is 0 or 1, which motifs are formed as aresult of the tertiary structure of said polypeptide. In relatedembodiments, wherein the amino acid motif comprises N₁ and N₂, thetertiary structures form such that the distance between the carbonylcarbons of N₁ and N₂ is about 3 Å to about 5 Å, preferably about 4 Å toabout 5 Å, and more preferably about 4.4 Å to about 4.8 Å. In otherembodiments, wherein the amino acid motif comprises N₁ and N₂, thetertiary structures form such that the distance between N₁ and N₂ areconfined spatially such that the charge separation, e.g., the chargedside chains, of the two is between about 6.5 Å to about 9 Å. In arelated embodiment, N₁ and N₂ are thus spatially confined as a result ofbeing in an amino acid sequence that forms all or a portion of an alphahelix, and may be separated by 1, 2, or more than 2 amino acids in thesequence of said amino acids forming said helix. In other relatedembodiments, wherein the amino acid motif comprises N₁ and P₁, thetertiary structures form such that the distance between the carbonylcarbons of N₁ and P₁ is about 3 Å to about 5 Å, preferably about 4 Å toabout 5 Å, and more preferably about 4.4 Å to about 4.8 Å. In otherembodiments, wherein the amino acid motif comprises N₁ and P₁, thetertiary structures form such that the distance between N₁ and P₁ areconfined spatially such that the charge separation, e.g., the chargedside chains, of the two is between about 6.5 Å to about 9 Å. In arelated embodiment, N₁ and P₁ are spatially confined as a result ofbeing in an amino acid sequence that forms all or a portion of an alphahelix, and may be separated by 1, 2, or more than 2 amino acids in thesequence of said amino acids forming said helix. In certain embodiments,the amino acids forming the motif within the tertiary structure of saidpolypeptide are separated from each other by an equal number ofintervening amino acid residues in the linear amino acid sequence ofsaid polypeptide. In yet other embodiments, the amino acids fowling themotif within the tertiary structure of said polypeptide are separatedfrom each other by a different number of intervening amino acid residuesin the linear amino acid sequence of said polypeptide. In certainembodiments, the isolated polypeptide of the inventions forms a regulartertiary structure, e.g., α-helix or β-pleated sheet, such that thesurface of said structure presents the amino acids comprising saidmotif, and thus the motif itself, to the interface of the proteinstructure and the aqueous environment, i.e., presents the motif on thesurface of folded the polypeptide. In preferred embodiments, thetertiary structures of the polypeptides of the invention form in anaqueous environment at physiological conditions, e.g., PBS (13 mMNaH₂PO₄, 137 mM NaCl, pH 7.4) at 37° C.

In specific embodiments, the invention provides for isolatedpolypeptides comprising the amino acid motifis described herein above,e.g., peptide A (APPRLICDSRVLERYLLEAKEAE, SEQ ID NO:32), peptide C(NITVPDTKVNFYAWKRMEVG, SEQ ID NO:29), peptide D(QQAVEVWQGLALLSEAVLRGQALLV, SEQ ID NO:30), peptide E(GCAEHCSLNENITVPDTKVN, SEQ ID NO:31), peptide F (RYLLUNITTGC, SEQ IDNO:33), peptide G (QEQLERALNSS, SEQ ID NO:40), peptide I(CSLNENIQEQLERALNSS, SEQ ID NO:43), peptide J (QEQLERALNSSLRRYINMLTRTR,SEQ ID NO:41), peptide K (WEHVNAIQEARRLL, SEQ ID NO:35), or peptide L(KIRSDLTALTESYVKH, SEQ ID NO:37).

In certain embodiments, the invention provides isolated polypeptidescomprising 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, or more than 6 amino acid motifs described herein. In specificaspects of the invention in accordance with this embodiment, wherein theisolated polypeptide comprises at least two of the amino acid motifsdescribed herein above, said at least two motifs may be the same motifor they may be different motifs.

In certain aspects, the invention provides for isolated polypeptideslacking an erythropoietic activity, e.g., increasing hemoglobin in arecipient. Preferably, the isolated polypeptides lack other activitiesincluding, but not limited to, vasoactive action (e.g.,vasoconstriction), hyperactivating platelets, pro-coagulant activitiesand stimulating proliferation and/or production of thrombocytes and/orerythropoietic-dependent cells (see, Coleman et al., 2006, PNAS103:5965-5970, hereby incorporated by reference in its entirety). Inother aspects, the invention provides isolated polypeptides thatcomprise at least one cellular protective activity. Such cellularprotective activity includes, but is not limited to, protecting,maintaining, enhancing or restoring the function or viability of aresponsive mammalian cell, tissue, or organ. Accordingly, in one aspect,the present invention is directed to the use of an isolated polypeptidedescribed herein for the preparation of pharmaceutical compositions forprotecting, maintaining, enhancing, or restoring the function orviability of responsive mammalian cells and their associated cells,tissues, and organs. In related embodiments, the compositions are foradministration to a subject in need thereof. In preferred embodiments,said subject is a mammal and, preferably, a human.

In other aspects, the present invention is directed to the use of anisolated polypeptide described herein for the preparation of apharmaceutical composition for the protection against and/or preventionof a responsive tissue injury, for the restoration of, or for therejuvenation of responsive tissue and/or responsive tissue function in asubject in need thereof. In one particular aspect, the responsivemammalian cells and their associated cells, tissues, or organs aredistal to the vasculature by virtue of a tight endothelial cell barrier.In another particular aspect, the cells, tissues, organs or other bodilyparts are isolated from a mammalian body, such as those intended fortransplant. By way of non-limiting examples, a responsive cell or tissuemay be neuronal, eye (e.g., retinal), adipose, connective, hair, teeth,mucosal, pancreas, endocrine, ear, epithelial, skin, muscle, heart,lung, liver, kidney, intestine, adrenal (e.g., adrenal cortex, adrenalmedulla), capillary, endothelial, testes, ovary, bone, skin, orendometrial cells or tissue. Further, non-limiting examples ofresponsive cells include photoreceptor (rods and cones), ganglion,bipolar, horizontal, amacrine, Müller, Purkinje, myocardium, pace maker,sinoatrial node, sinus node, junction tissue, atrioventricular node,bundle of His, hepatocytes, stellate, Kupffer, mesangial, renalepithelial, tubular interstitial, goblet, intestinal gland (crypts),enteral endocrine, glomerulosa, fasciculate, reticularis, chromaffin,pericyte, Leydig, Sertoli, sperm, Graffian follicle, primordialfollicle, islets of Langerhans, α-cells, β-cells, γ-cells, F-cells,osteoprogenitor, osteoclasts, osteoblasts, endometrial stroma,endometrial, stem and endothelial cells. These examples of responsivecells are merely illustrative. In one aspect, the responsive cell or itsassociated cells, tissues, or organs are excitable cells, tissues, ororgans, or predominantly comprise excitable cells or tissues. In certainaspects of the invention, the excitable tissue is central nervous systemtissue, peripheral nervous system tissue, cardiac tissue or retinaltissue. In another aspect, the responsive cell or its associated cells,tissues, or organs are not excitable cells, tissues, or organs, nor dothey predominantly comprise excitable cells or tissues.

The erythropoietic and/or cellular protective activity of the isolatedpolypeptide of the invention in responsive cells may be evaluated and/ordetermined by any method described herein and or known in the art. Incertain embodiments, the erythropoietic and/or cellular protectiveactivity is determined in an in vitro assay. In other embodiments, theerythropoietic and/or cellular protective activity is determined in anin vivo assay. In a related embodiment, wherein the cellular protectiveactivity is neuroprotection, the invention provides for a method ofevaluating said activity in vitro by (a) contacting a test culture ofprimary hippocampal neurons with N-methyl-D-aspartate and said peptide;and (b) determining the cell viability at 48 hours post said contact,such that if the cell viability determined in step (b) is greater thanthat of a control culture in the absence of said peptide, the peptidepossesses cellular protective activity.

In a particular embodiment, the mammalian cell, tissue, or organ forwhich an aforementioned isolated peptide is used are those that haveexpended or will expend a period of time under at least one conditionadverse to the viability of the cell, tissue, or organ. In accordancewith this embodiment, the isolated peptides of the invention provideprotection against and/or prevention of a tissue injury resulting fromsuch conditions, provide for the restoration of, or provide for therejuvenation of tissue and/or tissue function in a subject in needthereof before, during or after such conditions arise. Such conditionsinclude traumatic in situ hypoxia or metabolic dysfunction,surgically-induced in situ hypoxia or metabolic dysfunction, or in situtoxin exposure, the latter may be associated with chemotherapy orradiation therapy. In other embodiments, the isolated peptides of theinvention provide protection against and/or prevention of a tissueinjury resulting from a disease or disorder, provide for the restorationof, or provide for the rejuvenation of tissue and/or tissue function ina subject in need thereof before, during or after such conditions arise.In related embodiments said injury is caused by a seizure disorder,multiple sclerosis, stroke, hypotension, cardiac arrest, ischemia,myocardial infarction, inflammation, age-related loss of cognitivefunction, radiation damage, cerebral palsy, neurodegenerative disease,Alzheimer's disease, Parkinson's disease, mitochondrial disease, AIDSdementia, memory loss, amyotrophic lateral sclerosis, alcoholism, mooddisorder, anxiety disorder, attention deficit disorder, autism,Creutzfeld-Jakob disease, brain or spinal cord trauma or ischemia,heart-lung bypass, chronic heart failure, macular degeneration, diabeticneuropathy, diabetic retinopathy, hepatitis, pancreatitis, glaucoma,retinal ischemia, retinal trauma, cardiovascular disease,cardiopulmonary disease, respiratory disease, kidney disease, disease ofthe urinary system, disease of the reproductive system, bone disease,skin disease, connective tissue disease, gastrointestinal disease,endocrine abnormality, metabolic abnormality, or a disease or disorderof the central or peripheral nervous system. In still other embodiments,the adverse conditions are a result of cardio-pulmonary bypass(heart-lung machine), as is used for certain surgical procedures. Instill other embodiments, said injury is cognitive dysfunction. In aparticular embodiment, the mammalian cell, tissue, or organ for which anaforementioned isolated peptide is used express the βc receptor.

In certain embodiments, the invention is also directed to pharmaceuticalcompositions comprising the aforementioned isolated polypeptides foradministration to a subject in need thereof. In specific aspects inaccordance with this embodiment, the pharmaceutical composition of theinvention further comprises a pharmaceutically acceptable carrier. Suchpharmaceutical compositions may be formulated for oral, intranasal,ocular, inhalational, transdermal, rectal, sublingual, vaginal, orparenteral administration, or in the form of a perfusate solution formaintaining the viability of cells, tissues, or organs ex vivo. Inrelated embodiments of the invention the subject is a mammalian animal,preferably a human.

In other aspects, the invention provides a method for facilitating thetranscytosis of a molecule across an endothelial cell barrier in asubject in need thereof comprising administration to said subject acomposition comprising said molecule in association with an isolatedpeptide of the invention described hereinabove. In a related embodiment,association is a labile covalent bond, a stable covalent bond, or anon-covalent association with a binding site for said molecule.

According to another aspect of the invention, the isolated peptide ofthe invention, as described herein above, is capable of traversing anendothelial cell barrier. In a related embodiment, the endothelial cellbarrier comprises the blood-brain barrier, the blood-eye barrier, theblood-testis barrier, the blood-ovary barrier, blood-placenta,blood-heart, blood-kidney, blood-nerve, or blood-spinal cord barrier.

According to one aspect of the invention, there is provided an isolatednucleic acid molecule that comprises a nucleotide sequence which encodesa polypeptide comprising the isolated polypeptide as described hereinabove.

In another embodiment of the invention, there is provided an isolatednucleic acid molecule that comprises a nucleotide sequence (i.e., acDNA, a nucleotide sequence interrupted by introns, or uninterrupted bynitrous), which encodes a polypeptide comprising or consisting of theisolated polypeptide of the invention as described herein above. In oneembodiment, the nucleotide sequence, encoding the isolated polypeptideof the invention, is synthesized using preferred codons that facilitateoptimal expression in a particular host cell. Such preferred codons canbe optimal for expression in cells of a species of plant, bacterium,yeast, mammal, fungus, or insect.

The invention also provides for a vector comprising the nucleic acidmolecule. The invention also provides for an expression vectorcomprising the nucleic acid molecule and at least one regulatory regionoperably linked to the nucleic acid molecule. In another embodiment, theinvention provides for a cell comprising the expression vector. In yetanother embodiment, there is provided a genetically-engineered cellwhich comprises the nucleic acid molecule.

In another embodiment, the invention provides for a method ofrecombinantly producing the isolated peptide of the invention, describedherein above, comprising culturing in a medium a host cell containing anucleic acid molecule comprising a nucleotide sequence encoding apolypeptide of the invention, under conditions suitable for theexpression of said peptide, and recovering and/or isolating theexpressed polypeptide from said medium.

3.1 TERMINOLOGY

As used herein, the terms “about” or “approximately” when used inconjunction with a number refer to any number within 1, 5, or 10% of thereferenced number.

The term “administered in conjunction with” in the context of themethods of the invention means administering a compound prior to, at thesame time as, and/or subsequent to the onset of a disease, disorder, orcondition.

The term “amino acid” or any reference to a specific amino acid is meantto include naturally occurring proteogenic amino acids as well asnon-naturally occurring amino acids such as amino acid analogs. Thoseskilled in the art would know that this definition includes, unlessotherwise specifically noted, includes naturally occurring protogenic(L)-amino acids, their optical (D)-isomers, chemically modified aminoacids, including amino acid analogs such as penicillamine(3-mercapto-D-valine), naturally occurring non-proteogenic amino acidssuch as norleucine and chemically synthesized proteins that haveproperties known in the art to be characteristic of an amino acid. Asused herein, amino acids will be represented wither by their threeletter acronym or one letter symbol as follows: alanine=Ala or A,arginine=Arg or R, asparagine=Asn or N, aspartic acid=Asp or D, cysteineCys or C, glutamic acid=Glu or E, glutamine=Gln or Q, glycine=Gly or G,histidine=His or H, isoleucine=Ile or I, leucine=Leu or L, lysine=Lys orK, methionine=Met or M, phenylalanine Phe or F, proline=Pro or P,serine=Ser or S, threonine=Thr or T, tryptophan=Trp or W, tyrosine=Tyror Y, and valine=Val or V. Additionally, the term “amino acidequivalent” refers to compounds that depart from the structure of thenaturally occurring amino acids, but which have substantially thestructure of an amino acid, such that they can be substituted within apeptide, which retains its biological activity despite the substitution.Thus, for example, amino acid equivalents can include amino acids havingside chain modifications or substitutions, and also include relatedorganic acids, amides or the like. The term “amino acid” is intended toinclude amino acid equivalents. The term “residues” refers both to aminoacids and amino acid equivalents. Amino acids may also be classifiedinto the following groups as is commonly known in the art: (1)hydrophobic amino acids: His, Trp, Tyr, Phe, Met, Leu, Ile, Val, Ala;(2) neutral hydrophilic amino acids: Cys, Ser, Thr; (3) polar aminoacids: Ser, Thr, Asn, Gln; (4) acidic/negatively charged amino acids:Asp, Glu; (5) charged amino acids: Asp, Glu, Arg, Lys, His; (6)positively charged amino acids: Arg, Lys, His; and (7) basic aminoacids: His, Lys, Arg.

As used herein, “excitable tissue” means tissue that contains excitablecells. Excitable cells are cells that respond actively to an electricstimulus and have an electrical charge differential across theircellular membranes. Excitable cells are generally capable of undergoingan action potential. Such cells typically express channels, such asvoltage-gated, ligand-gated, and stretch channels, which allow flow ofions (potassium, sodium, calcium, chloride, etc.) across the membrane.Excitable tissue includes neuronal tissue, muscle tissue, and glandulartissue. Excitable tissue includes, but is not limited to, neuronaltissues such as tissue of the peripheral nervous system (ear and retina)and central nervous system (brain and spinal cord); cardiovasculartissue such as the cells of the heart and associated nerves; andglandular tissue such as the pancreas where T-type calcium channelsalong with cell-to-cell gap junctions participate in secretion ofinsulin. An exemplary list of excitable tissue includes organs andtissues that include nerves, skeletal muscle, smooth muscle, cardiacmuscle, uterus, central nervous system, spinal cord, brain, retina,olfactory system, auditory system, etc.

The term “host cell” as used herein refers to the particular subjectcell transfected with a nucleic acid molecule and the progeny orpotential progeny of such a cell. Progeny of such a cell may not beidentical to the parent cell transfected with the nucleic acid moleculedue to mutations or environmental influences that may occur insucceeding generations or integration of the nucleic acid molecule intothe host cell genome.

An “isolated” or “purified” polypeptide is substantially free ofcellular material or other contaminating proteins from the cell ortissue source from which the protein or polypeptide is derived, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of a polypeptide in which thepolypeptide is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. Thus, a polypeptide thatis substantially free of cellular material includes preparations ofpolypeptides having less than about 30%, 20%, 10%, or 5% (by dry weight)of heterologous protein (also referred to herein as a “contaminatingprotein”). When the polypeptide is recombinantly produced, it is alsopreferably substantially free of culture medium, i.e., culture mediumrepresents less than about 20%, 10%, or 5% of the volume of the proteinpreparation. When the polypeptide is produced by chemical synthesis, itis preferably substantially free of chemical precursors or otherchemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein.Accordingly such preparations of the polypeptide have less than about30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compoundsother than the antibody of interest. In a preferred embodiment,polypeptides of the invention are isolated or purified.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. In a specific embodiment, a nucleic acidmolecule(s) encoding a polypeptide of the invention is isolated orpurified.

As used herein in reference to a structure within a polypeptide, theterm “motif” refers either to a set of consecutive amino acids withinthe amino acid sequence of the polypeptide chain and/or to a set oflinearly adjacent amino acids within the tertiary structure of saidpolypeptide. Because the motif may be formed all or in part as a resultof protein folding, amino acids that are adjacent in the described motifmay be separated by 0, 1 or more, 5 or more, 10 or more, 15 or more or20 or more amino acids within the linear amino acid sequence of thepolypeptide.

As used herein, the terms “peptide,” “polypeptide” d “protein” are usedinterchangeably and in their broadest sense to refer to constrained(that is, having some element of structure as, for example, the presenceof amino acids which initiate a β turn or β pleated sheet, or forexample, cyclized by the presence of disulfide bonded Cys residues) orunconstrained (e.g., linear) amino acid sequences. In certainembodiments, the peptide of the invention consists of less than 30 aminoacids. However, upon reading the instant disclosure, the skilled artisanwill recognize that it is not the length of a particular peptide but itsability to bind a tissue protective receptor complex and/or compete withthe binding of a peptide described herein that distinguishes the peptideof the invention. The terms “peptide,” “polypeptide,” d “protein” alsorefer to compounds containing amino acid equivalents or other non-aminoacid groups, while still retaining the desired functional activity of apeptide. Peptide equivalents can differ from conventional peptides bythe replacement of one or more amino acids with related organic acids(such as PABA), amino acids or the like or the substitution ormodification of side chains or functional groups.

The term “preventing a disease, disorder, or condition” means delayingthe onset, hindering the progress, hindering the appearance, protectionagainst, inhibiting or eliminating the emergence, or reducing theincidence, of such disease, disorder, or condition. Use of the term“prevention” is not meant to imply that all patients in a patientpopulation administered a preventative therapy will never develop thedisease, disorder, or condition targeted for prevention, but rather thatthe patient population will exhibit a reduction in the incidence of thedisease, disorder, or condition. For example, many flu vaccines are not100% effective at preventing flu in those administered the vaccine. Oneskilled in the art can readily identify patients and situations for whompreventative therapy would be beneficial, such as, but not limited to,individuals about to engage in activities that may lead to trauma andinjury (e.g., soldiers engaging in military operations, race cardrivers, etc.), patients for whom surgery is planned, patients at riskfor inherited diseases, disorders, or conditions, patients at risk fordiseases, disorders, or conditions precipitated by environmentalfactors, or portions of the population at risk for particular diseases,disorders, or conditions such as the elderly, infants, or those withweakened immune systems, or those patients with genetic or other riskfactors for a disease, disorder, or condition.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the terms “subject” and “subjects”refer to an animal, preferably a mammal including a non-primate (e.g., acow, pig, horse, cat, dog, rat, and mouse) and a non-primate (e.g., amonkey or a human), and more preferably a human.

As used herein, the term “tissue protective activity” or “tissueprotection” refers to the effect of inhibiting or delaying damage ordeath of a cell, tissue, or organ. Unless otherwise noted, the “delay”in damage or death of a cell, tissue or organ is evaluated relative to acontrol condition in the absence of a peptide of the invention. Thetissue protective activity is useful in various conditions, diseases,and cellular, organ, and/or tissue damage, for example, those describedin section 5.3. Tissue protective activity is specific to tissue, cells,and/or organs expressing a tissue protective receptor complex (i.e., aresponsive tissue cell, and.or organ, respectively), such as, but notlimited to, the tissues of the central nervous system. In specificembodiments, the responsive cells are not erythrocyte progenitor cells.

The term “tissue protective receptor complex” as used herein means acomplex comprising at least one erythropoietin receptor subunit and atleast one beta common receptor subunit. The tissue protective receptorcomplex may contain multiple erythropoietin receptor subunits and/orbeta common receptor subunits, as well as other types of receptors orproteins. See WO 2004/096148, which is hereby incorporated by referenceherein in its entirety.

To determine the percent identity of two amino acid sequences, thesequences are aligned for optimal comparison purposes. The amino acidresidues at corresponding amino acid positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue as the corresponding position in the second sequence, then themolecules are identical at that position. The percent identity betweenthe two sequences is a function of the number of identical positionsshared by the sequences (i.e., % identity=number of identicaloverlapping positions/total number of positions×100%). In oneembodiment, the two sequences are the same length. In an alternateembodiment, the sequences are of different length and, accordingly, thepercent identity refers to a comparison of the shorter sequence to aportion of the longer sequence, wherein said portion is the same lengthas said shorter sequence.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the results of an in vivo sciatic nerve injury model tocompare the efficacy of peptide J (SEQ ID NO:41) to the tissueprotective molecule carbamylated EPO (CEPO), wherein peptide J, SEQ IDNO:41, is a chimeric peptide consisting of the external facing aminoacids of helix B of EPO (i.e., peptide G, SEQ ID NO:40) combined with anamphipathic helix from pancreatic polypeptide (LRRYINMLTRP, SEQ IDNO:28)

FIG. 2 depicts the tissue protective effects of peptides of theinvention as tested in an in vivo sciatic nerve injury model. In theassay, the right sciatic nerve of rats (n=6 per group) was injured andthe animal immediately dosed with PBS, or PBS containing equal molarconcentrations of carbamylated EPO, EPO peptide A (SEQ ID NO:32,corresponding to amino acids 1-23 of SEQ NO:1), peptide D (SEQ ID NO:30,corresponding to amino acids 58-82 of SEQ ID NO:1), or peptide G (SEQ IDNO:40). Peptide G (SEQ ID NO:40) is based on those amino acids withinHelix B of EPO that face outward from the globular center of the EPOmolecule into the hydrophilic environment, i.e., present on the surfaceof the polypeptide. Additionally, a 20-mer constructed from a region ofpigment epithelium-derived factor known to be tissue protective viaanother receptor was included as a negative control. The recovery frominjury over the next 4 days demonstrates that peptide G, SEQ ID NO:40,and peptide D, SEQ ID NO:30, exhibit a tissue protective effect in thisin vivo model assay that is equivalent to or better than carbamylatedEPO (CEPO).

FIG. 3 depicts the erythropoietic effects of peptide D, SEQ ID NO:30,and CEPO, known to lack erythropoietic activity, as tested in a UT-7assay for erythropoietic activity. The results of this in vitro assaydemonstrate that neither peptide D, SEQ ID NO:30, nor CEPO exhibiterythropoietic activity at doses up to 10,000 pM.

FIG. 4 depicts the results of an in vivo assay to determine whetherpeptide F (SEQ ID NO:33, corresponding to amino acids 14-29 of SEQ IDNO:1) and peptide G (SEQ NO:40) are erythropoietic or elicitneutralizing antibodies against EPO. The results demonstrate thatneither protein increased hemoglobin levels in the rats whenadministered at 0.8 μg/kg, 3 days/week sub-cutaneously (s.c.) over thecourse of 130 days. In addition, neither peptide elicits an antibodyresponse, in contrast to the administration of EPO.

FIG. 5 depicts the results of in vitro studies that demonstrate thatpeptide D, SEQ ID NO:30, protects motor neurons against kainate induceddeath.

FIG. 6 shows that peptide D, SEQ NO:30, at doses of 0.1 ng/ml and 1ng/ml protects P-19 cells against apoptosis associated with serumdeprivation.

FIGS. 7 A-B depict the results of a middle cerebral artery occlusionassay in rats. FIG. 7A depicts a graph demonstrating that peptide D (SEQID NO:30, corresponding to amino acids 58-82 of SEQ ID NO:1) at a singledose of 4.4 ug/kg is able to reduce the volume of the infarct in thebrain as robustly as four doses of 4.4 ug/kg administered 2 hours apart.FIG. 7B depicts the results of a foot fault assay to determine thebehavioral deficit caused by the middle cerebral artery occlusion. FIG.7B shows that rats demonstrated behavioral improvements whenadministered peptide D, SEQ NO:30, at both a single dose schedule (1×4.4ug/kg) and a multiple dose schedule (4×4.4 ug/kg).

FIGS. 8 A-B depict the results of an in vivo assay of a diabeticneuropathy assay. Diabetes is induced in rats using streptozotocin.After verification of induced diabetes, the rats were treated withpeptide D, SEQ ID NO:30, or PBS five times a week at a dose of 4ug/kg-bw i.p. for a period of two weeks. Both the nerve conductionvelocity and the hot plate latency of the rats were observed. FIG. 8Ademonstrates that the rats treated with peptide D, SEQ ID NO:30exhibited improved conduction velocities in comparison to the untreatedrats. FIG. 8B demonstrates that hotplate latency for the treated ratswas reduced relative to the untreated rats, further demonstrating theimprovement in conduction velocity.

FIGS. 9 A-B depict the results of treatment of cisplatin inducedneuropathy and nephropathy with EPO Helix B chimera. FIG. 9Ademonstrates that the animals treated with peptide G (SEQ ID NO:40, aHelix B chimera) exhibited improved results when tested in a hotplatelatency assay. FIG. 9B demonstrates that the urine production, a measureof kidney function, was maintained as normal in the peptide G (SEQ IDNO:40) treated animals.

FIG. 10 depicts the effects of peptide D (SEQ ID NO:30) on retinalleakage associated with diabetic retinopathy. The figure demonstratesthat peptide D (SEQ ID NO:30) was able to substantially reduce retinalleakage in the treated animals.

FIG. 11 depicts the results of peptide F (SEQ ID NO:33) or peptide G(SEQ ID NO:40) on a model of kidney ischemia-reperfusion. The figuredemonstrates that both peptides reduced the injury score resulting froman ischemia-reperfusion injury of 60 minutes when assessed after 72hours

FIG. 12 illustrates that the administration of peptide F (SEQ ID NO:33)protects mice from experimental cerebral malaria.

FIG. 13 Clinical Score in murine EAE model treated with Peptide E, SEQID NO:31. FIG. 13 depicts the clinical course of neurological functionin mice with experimental autoimmune encephalomyelitis. 4.4 ug/kgPeptide E was administered i.p. daily. Administration of peptide Esignificantly improved neurological function relative to control.Clinical staging; 1, flaccid tail; 2, ataxia and/or hind-limb paresis,or slow righting reflex; 3, paralysis of hind limb and/or paresis offorelimbs; 4, paresis of forelimb; 5, moribund or death.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 Tissue Protective Peptides

The erythropoietic activity of erythropoietin (“EPO”) has been wellcharacterized in the art (see, e.g., Cheetham et al., 1998, Nat. Struct.Biol. 5; 861-866, herein incorporated by reference in its entirety). EPOinitiates erythropoiesis by binding to the extracellular portion of apreformed erythropoietin receptor (EPOR) homodimer (i.e., (EPOR)₂) in amanner that bridges between specific locations on the individual EPORsubunits. When EPO binds to the (EPOR)₂, large portions of the globularligand are remote from the binding regions and face outward, away fromthe complex of EPO and (EPOR)₂ into the aqueous medium. The Applicantshave determined that tissue protection, as distinct from erythropoiesis,is mediated through a receptor other than (EPOR)₂, which consists of anEPOR monomer in conjunction with another receptor, CD131 (also known asthe β-common receptor subunit (β_(c))). EPOR and β_(c) interact to formthe receptor heterodimer, EPOR-β_(c). Whether other proteins areinvolved in this interaction is currently unknown. The instant inventiondiscloses tissue protective peptides derived from the three dimensionalstructure of EPO, and in particular, from the portions of EPO facingaway from the EPOR binding sites, i.e., not interacting with, theclassical, erythropoietic EPOR (EPOR)₂ homodimer. Not wishing to bebound by any particular theory, the Applicants believe that this portionof the EPO molecule interacts with the tissue protective receptor andthereby mediates tissue protection.

The three dimensional structure of EPO is accepted as described byCheetham et al., 1998, Nat. Struct. Biol. 5; 861-866, herebyincorporated by reference in its entirety, and as set forth in SEQ IDNO:1 (also available as data deposited in the Protein Data Bank of theNational Center for Biotechnology Information as entry “1BUY”). Theportions of the EPO molecule that face away from the membrane-proximalportion of the EPOR homodimer when bound to said receptor (i.e., awayfrom the cell membrane when the (EPOR)₂ homodimer is expressed on thesurface of a cell) consist of the following secondary structures: loopAB (corresponding to amino acids 29-55 of SEQ ID NO:1), helix B(corresponding to amino acids 56-82 of SEQ ID NO:1), loop BC(corresponding to amino acids 83-92 of SEQ ID NO:1) and loop CD(corresponding to amino acids 112-138 of SEQ ID NO:1). In one embodimentof the invention, the tissue protective peptides consist of the aminoacid sequences corresponding to these distinct structures of the EPOmolecule.

Not wishing to be bound to any particular theory, the Applicants believethat the Tissue Protective Receptor is preformed, i.e. that the EPOR andβ_(c) protein subunits are functionally associated prior to theirinteraction with EPO. EPO is a member of the type I cytokinesuperfamily. Members of type 1 cytokine superfamily branch arecharacterized by four helices which interact hydrophobically to form aglobular protein whose exterior surface interfaces with the aqueousmedium and is termed “externally-facing”. Unexpectedly, the Applicantshave determined that more than one peptide derived from theexternally-facing portion of the EPO molecule is tissue-protective. Afurther surprising discovery is that peptides derived from portions ofthe EPO molecule that are buried within the EPO:(EPOR)₂ complex andpeptides that may also contain portions of erythropoiesis binding sites1 or 2 are also be highly potent in tissue protection. To account forthese discoveries, Applicants propose that successful activation of thetissue protective receptor is due to an appropriate, spatially compactcharge configuration within the peptide ligand. Further, this compactcharge configuration is embodied by two distinct structural motifs: (1)two negatively charged amino acids adjacent to each other, and flankedby hydrophobic amino acids; or (2) a positive and a negative (i.e.,basic and acidic) amino acid immediately adjacent to one another, andflanked by single hydrophobic or polar amino acid residues. Theproximity of these charges may occur via the linear structure imposed bypeptide bonding, i.e., the structure may be formed by consecutive aminoacids in a polypeptide chain, or alternatively, proximity can also occurvia a spatial relationship between different parts of the EPO molecule(or other related type 1 cytokine molecules) imparted by the protein'stertiary structure, i.e., three dimensional structure. Not wishing to bebound to any specific theory, Applicants believe that, in general, thisrequirement dictates that a tissue protective peptide will have adistinct tertiary structure (e.g., helices or pleated sheets) thatprovides for the required spatial location of the pair of charged aminoacids (i.e., the two negatively charges amino acids and/or the positiveand negative amino acid). A simple exception is a linear peptide whereinthe amino acid pair is immediately adjacent to each other, with therequired rigidity imparted by the peptide backbone. Accordingly, thestructural motif (1), is encompassed by a linear sequence of amino acidresidues, e.g., H₁—N₁—N₁—H₂(SEQ ID NO:6), or by a linear sequence ofamino acid residues wherein N₁ and N₂ are separated by 1, 2, 3, 4, 5, 6,or more intervening residues, e.g., H₁—N₁—X—X—X—X—X—N₁—H₂ (SEQ IDNO:11).

For tissue protection, the pair of charged amino acids must be spatiallyoriented such that the carbonyl carbons are about 3 angstroms (Å) toabout 5 Å apart, preferably, about 4 Å to about 5 Å apart, and morepreferably about 4.4 Å to about 4.8 Å apart. This can be accomplished ina number of ways, for example, by adjacent charged amino acids in asimple linear peptide (see, e.g., Example 2 and peptide G, SEQ ID NO:40,Table 1) or for peptides that can form an alpha helix, charged aminoacids separated by an intervening amino acid residue (see, e.g., Example2 and peptide F, SEQ ID NO:33, Table 1). It is to be noted that tertiarystructure (e.g., an alpha helix in amphipathic peptides) can also beimparted when the peptide is within a specific microenvironment, such asat the extracellular-cell surface membrane interface (see, Segrest,1990, Proteins 8:103-117, hereby incorporated by reference in itsentirety).

Further, tissue protective activity is predicted for peptides thatcontain pairs of charged amino acids such that the charged side-chains(either positive and negative or two negatives) be confined spatially towithin about 6.5 Å to about 9 Å of each other. This can be provided forin an alpha helix by the charged pair being separated by one or twoamino acids, which will provide for the charges to be more or less onthe same side of the helix with the required about 6.5 Å to about 9 Åseparation. A non-limiting example of such a peptide is found in peptideF (see, Example 2, SEQ ID NO:33, Table 1). One skilled in the art candevise a tertiary structure for the peptide that is generally requiredto obtain the appropriate three dimensional location of the chargedamino acids, as well as the design of small molecules to mimic thecharge separation within the peptide.

The spatial distances between the carbamyl carbons of any to amino acidsor between the side chains of any two amino acids can be deduced by anymethod known in the art or described herein. For example, where thethree-dimensional structure of the protein is known, the chargeseparation of two side chains or the spatial distance between twocarbamyl carbons within a portion of interest of said protein can becalculated based on the published, or otherwise art-accepted,three-dimensional coordinates of the amino acid residues in said portionof interest. Where the three-dimensional structure of the protein and,therefore, the portion of interest is unknown, or wherein a fullysynthetic peptide is constructed based on the teachings herein, whosethree dimensional structure is unknown, the charge separation of twoside chains or the spatial distance between two carbamyl carbons withinsaid peptide can be estimated using the three-dimensional structurepredicted by protein modeling software as is known in the art.Non-limiting examples of such software are MOE™ by Chemical ComputingGroup (Quebec, Canada) and Modeler by Accelrys (San Diego, Calif.).Similarly such predictive software, available from the above-notedcompanies as well, is also known in the art for the design of smallmolecules as and, accordingly, one of ordinary skill in the art, basedupon the teachings herein, would be able to make small molecules thatemulate the disclosed structural motifs.

Non-naturally occurring or chimeric peptides can be designed that mimicthe critical spatial proximities described herein above via a linearsequence of amino acids. The present invention is, therefore, directedto novel tissue protective peptides, including those that exhibit thesestructural motifs that trigger tissue protection.

The present invention also relates to the use of tissue protectivefragments of other type 1 cytokines, including, but not limited to,granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3(IL-3), Thrombopoietin (TPO), Ciliary Neurotrophic Factor (CNTF) andLeukemia Inhibitory Factor (LIF), that are structurally homologous withthe above noted externally-presenting amino acid sequences of EPO and/orcontain the structural motifs described above.

Further, the tissue protective peptides may be chimeric compounds basedupon structural motifs described above combining non-adjacent structuralelements and surface presenting amino acids solely. In particular, theapplicants have determined that the addition of an amphipathic peptidehelix to the above noted sequences increases the potency of the peptide.

Additionally, the tissue protective peptides of the present inventioninclude fusion peptides resulting from the combination of two or more ofthe above noted peptides, or with a related or unrelated macromoleculefor specific transport, such as native EPO, insulin or leptin.

5.1.1 Fragments A. EPO-derived Peptide Fragments

The present invention relates to novel tissue protective peptides thatin one embodiment are comprised of fragments of the amino acid sequencesof EPO, derived from the three dimensional structure of the EPO protein,and in particular, were derived from those regions of EPO facing awayfrom the ligand binding sites and/or the internal portion of the EPORhomodimer. These fragments are derived from the following EPOstructures: (1) loop AB and N-terminal portion of helix B(NITVPDTKVNFYAWKRMEVG, SEQ ID NO:29, corresponding to amino acids 38-57of SEQ ID NO:1); (2) C-terminal portion of helix B(QQAVEVWQGLALLSEAVLRGQALLV, SEQ ID NO:30, corresponding to amino acids58-82 of SEQ ID NO:1), and (3) a portion of the A-B loop consisting of asmall cysteine loop and a β-pleated sheet (GCAEHCSLNENITVPDTKVN, SEQ IDNO:31, corresponding to amino acids 28-47 of SEQ ID NO:1). These peptidefragments are all demonstrated in Example 2 (see FIG. 1 and Table 1) toexhibit tissue protective properties.

Unexpectedly, some peptides derived from other regions of the EPOmolecule that are buried and other peptides that include portions of thebinding sites to (EPOR)₂ are also tissue protective. For example, apeptide consisting of the N-terminal portion of Helix A(APPRLICDSRVLERYLLEAKEAE, SEQ ID NO:32, corresponding to amino acids1-23 of SEQ ID NO:1) that contains a portion of EPOR binding Site 2(underlined) is tissue protective (see Example 2 and Table 1). However,the presence of Site 2 amino acids does not account for the tissueprotective activity, as a peptide consisting of amino acids 14-19 of SEQID NO:1 (RYLLEAKEAENITTGC, SEQ ID NO:33) and lacking amino acids 11-13of SEQ ID NO:1 (i.e., VLE; the site 2 amino acids that are required forbinding of EPO to the EPOR dimer, (EPOR)₂, is also tissue protective(see, Example 2 and Table 1, also Elliott et al., 1997, Blood 89:493,hereby incorporated by reference in its entirety). Applicants havepreviously shown that mutations within the erythropoiesis binding sitesthat abolish erythropoiesis do not modify the tissue protectiveproperties of EPO (Leist et al. Science (2004) 305:239, herebyincorporated by reference in its entirety).

One of ordinary skill in the art will recognize that fragments ofvarying lengths can form a tissue protective peptide, although thefragment is preferably less than 30 amino acids in length. Further,judicious selection of other molecules for inclusion, e.g., D-aminoacids or polyethylene glycol, will also constitute a tissue protectivepeptides, but with enhanced biological half-lives.

A. Structural Motifs

Specifically, the following structural motifs have been identified thattrigger the Tissue Protective Receptor complex:

(a) A Negative Charge Configuration (“Structural Motif A”)

In this structural motif, the peptide possesses two negatively chargedamino acids, which can be separated by up to 5 amino acids, flanked byhydrophobic amino acids. Structurally this can be represented as:

(a1) HNNH;

(a2) HNXNH;

(a3) HNXXNH;

(a4) HNXXXNH;

(a5) HNXXXXNH; or

(a6) HNXXXXXNH,

where H represents hydrophobic amino acids (e.g., the moderatelyhydrophobic amino acids: glycine, proline, cysteine, tyrosine, andtryptophan, and preferably the highly hydrophobic amino acids: alanine,valine, isoleucine, methionine, leucine, phenylalanine), N represents anegatively charged amino acid such as glutamate or aspartate, and Xrepresents any amino acid, although preferably a hydrophilic one. Incertain embodiments, the flanking hydrophobic amino acids are the same.In other embodiments, the flanking amino acids are different.

A variation of this structural motif involves a peptide where one of theflanking hydrophobic amino acids has been replaced with a polar aminoacid such as serine, threonine, asparagine, or glutamine.

As an alternative to peptide linkages establishing the mutual proximityof the two negative charges in a linear sequence, the necessary chargeproximity may also be accomplished by a three dimensional structure asdiscussed herein above, (Section 5.1). For example, the negativelycharged amino acids may be spatially immediately adjacent on theexternal surface of a helix, but will be separated by additional aminoacids in the linear peptide sequence. For example, in helix A of EPO(corresponding to amino acids 10-28 of SEQ ID NO:1), E18 and E21 areadjacent on the three dimensional structure, but have two interveningamino acids between them in the linear peptide sequence. As anadditional example, in helix B (peptide D, SEQ ID NO:30; correspondingto amino acids 58-82 of SEQ ID NO:1) E62 and E72 are separated by twoamino acids (Q65 and L69) on the surface of the helix, but have 9 aminoacids between them within the linear peptide. Peptides constructed fromhelix A or helix B are tissue protective (See Example 2 and Table 1,infra). In contrast, peptide B (NITTGCAEHCSLNE SEQ ID NO:34) a peptidewith dual negative charges (underlined) at the appropriate distance butlacking a flanking hydrophobic amino acid, is not tissue protective (SeeExample 2 and Table 1, infra).

(b) Negative/Positive Amino Acid Configuration (“Structural Motif B”).

In this structural motif, the peptide has a positive amino acid next toa negative amino acid and both charged amino acids are flanked by singlehydrophobic amino acids. Structurally this can be represented as:

(b1) HNPH; or

(b2) HPNH,

where P represents positively charged amino acids such as arginine,lysine or histidine and N represents the negatively charged amino acidsglutamate or aspartate. As with the first motif, the mutual proximity ofthe two opposite charges may be accomplished by three dimensionalstructure. For example, a positive and a negatively charged amino acidmay be spatially adjacent on the surface of a helix, but will beseparated by one or more amino acids in the linear peptide sequence. Forexample, in helix B (corresponding to amino acids 58-82 of SEQ ID NO:1)E72 and R76 are immediately adjacent to each other on the externalsurface of the helix and a peptide constructed from this helix is tissueprotective (see Example 2 and Table 1).

In a variation of this particular motif, the negative and positive aminoacids can be separated by a polar amino acid, e.g.,

(b3) HNLPH;

(b4) HPLNH,

wherein L represents a polar amino acids such as serine, threonine,asparagine, or Glutamine. An example of this motif is peptide E(GCAEHCSLNENITVPDTKVN, SEQ ID NO:34), which is tissue protective (seeExample 2 and Table 1).

Given that the core of the above structural motif is four amino acids inlength, a peptide of this core structural motif may trigger the TissueProtective Receptor. In certain embodiments the polypeptides of theinvention comprise 1 structural motif. In alternate embodiments, thepolypeptides of the invention comprise more than 1, more than 2, morethan 3 or more than 4 of the structural motifs. In certain embodiments,wherein the polypeptide comprises at least two structural motifs, themotifs are the same. In alternate embodiments, wherein the polypeptidecomprises at least two structural motifs, the motifs are different.Preferably, the multiple peptides of the present invention that oneskilled in the art can generate are less than 30 amino acids in length.

One of ordinary skill in the art will recognize that it is the abovenoted structural motifs, as opposed to the actual amino acid sequence ofEPO that is important to the current invention. Thus one of ordinaryskill in the art would recognize that the isolated peptide may have lessthan 90%, less than 85%, less than 80%, less than 75%, less than 70%,less than 65%, less than 60%, less than 55%, less than 50%, less than45%, less than 40%, less than 35%, less than 30%, or less than 20percent sequence identity with any portion of the amino acid sequence ofmature human erythropoietin (“EPO”) set forth in SEQ ID NO:1, whereinsaid portion of EPO contains the same number of amino acid residues assaid peptide.

Additionally, U.S. Pat. No. 5,700,909 to O'Brien et al., herebyincorporated by reference in its entirety) discloses a 17 amino acidpeptide sequence of EPO (SEQ ID NO:11 of O'Brien) which inducesbiological activity in NS20Y, SK-N-MC, and PC12 cells includingsprouting, differentiation, neuroprotection, and prevention of neuronalcell death. SEQ ID NO:11 of O'Brien (designed epopeptide AB), althoughprophetically, disclosed to have erythropoietic activity, in fact lackssuch erythropoietic activity and was subsequently found to lack in vivoactivity. When epopeptide AB was injected into the muscle of mice, thefrequency of motor end plate sprouting in the adjacent muscles increasedin a manner similar to that induced by ciliary neurotrophic factor.These data are interpreted within the concept that neuronal (but nothematological) cells respond to a peptide sequence within EPO and thatEPO may have separate domains for neurotrophic and hematotrophicactivity (Campana et al., Int. J. Mol. Med. (1998) 1(1):235-241; J. S.O'Brien in U.S. Pat. No. 5,700,909, issued Dec. 23, 1997; J. S. O'Brienin U.S. Pat. No. 5,571,787, issued Nov. 5, 1996; J. S. O'Brien in U.S.Pat. No. 5,714,459, issued Feb. 3, 1998; and J. S. O'Brien and Y.Kashimoto in U.S. Pat. No. 5,696,080, issued Dec. 9, 1997). However,O'Brien did not appreciate the current structural motifs based upon theproximity of charged amino acids in the tertiary structure of thepeptide.

C. Type 1 Cytokine Fragments.

Given the spatially compact charge configuration able to activate thetissue protective receptor, Applicants have discovered that certainfragments of type-1 cytokines are expected to cross react with thetissue protective receptor. This cytokine family includes, but is notlimited to, interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9,IL-10, IL-11, granulocyte macrophage-colony stimulating factor (GM-CSF),leptin, granulocyte colony stimulating factor (G-CSF), leukemiainhibiting factor (LIF), ciliary neurotrophic factor (CNTF),thrombopoietin (TPO), growth hormone, macrophage colony stimulatingfactor (M-CSF), erythropoietin (EPO) and prolactin.

Consideration of the secondary structure of EPO provides guidance forthe preparation of a candidate tissue protective peptide via the spatialarrangement of amino acids derived from homologous amino acids locatedwithin homologous secondary structures within other type-1 cytokinereceptor ligands: e.g., GM-CSF and IL-3 (Kannan, 2000, Neuroimmunomod.8:132-141, herby incorporated by reference in its entirety), amongothers, have been shown to possess potent neurotrophic andneuroprotective activities, due in large part, the Applicants believe,by stimulating a tissue protective receptor. For example, consideringhelix B of these type I cytokines: Homologous amino acids inthrombopoietin (TPO; Protein Data Bank (PDB) accession 1V7M) compriseD62, G65, T68, L69, E72, A76 and Q80, where these amino acids arespatially adjacent to one another in a linear arrangement; homologousamino acids in leukemia inhibitory factor (LIF; PDB accession 1EMR)comprise E61, R64, Y68, S72, N75, and D79; homologous amino acids inciliary neurotrophic factor (CNTF; PDB accession 1(NT) comprise E71,E75. These all are examples of Motif A described above section 5.1.1),wherein the underlined amino acids are negatively charged.

Examples of peptides derived from the Type-1 cytokines that exemplifythe structural Motif B described herein above (section 5.1.1) include,but are not limited to, GM-CSF helix A fragment, WEHVNAIQEARRLL (SEQ IDNO:35); TPO helix A fragment, LSKLLRDSHVLH (SEQ ID NO:36); TPO helix Bfragment: E56, K59; CNTF helix A fragment, KIRSDLTALTESYVKH (SEQ IDNO:37); CNTF helix B fragment: R89, E92. LIP helix B fragment,GTEKAKLVELYRIVVYL (SEQ ID NO:38); and interleukin 3 (IL-3) helix Afragment SIMIDEIIHHLKRPPNPL (SEQ ID NO:39).

These aforementioned amino acids are merely exemplary from some membersof the cytokine superfamily that signal through Type 1 cytokinereceptors, and homologous regions on other members of the cytokinesuperfamily will be readily identified by the skilled artisan.

5.1.2 Chimeras

“Chimeric” tissue protective peptides—linear amino acid sequences thatincorporate non-linear structural elements of the externally-facingamino acids of EPO molecule exhibit the above-noted structuralmotifs—are also contemplated by the current invention. Chimeric tissueprotective peptides of the current invention may consist of combiningstructural elements of separate amino acid sequences into a singlepeptide. In other words, a chimeric tissue protective peptide may becomprised of amino acid sequences derived from non-linear but adjacentstructural elements such as a fragment derived from amino acid sequences110-115, 133-136, and 160-165, of SEQ ID NO:1 which would allowstructural elements of the C terminal portion of helix C and N-terminalportion of loop C-D, the O-pleated sheet in loop C-D, and the C-terminalportion of EPO to be contained in a single peptide. Additionally,chimeric tissue protective peptides may be used to select out theimportant features of a particular structure, for example theexternally-facing amino acids of a particular tertiary structure. Thus,a chimeric tissue protective peptide may consist of a fragment comprisedof helix B amino acids 58, 62, 65, 69, 72, 76, 79, 80, 83, 84, and 85(e.g., peptide G, QEQLERALNSS, SEQ ID NO:40) or, in other words, all ofthe exterior-presenting amino acids of helix B of EPO. This peptide isshown to be tissue protective in Example 2, infra (see Table 1).

Furthermore, the potency of the current tissue protective peptides maybe increased by attaching an amphipathic peptide helix. Amphipathicpeptide helices are well known in the art, e.g. from peptides thatsignal through the Class B G-protein coupled receptors (e.g., Segrest etal., 1990, Proteins 8:103, hereby incorporated by reference in itsentirety), serving to localize the peptide ligand to the cell membrane.Examples of such helices include, but are not limited to, the highlyhydrophobic regions from: calcitonin (ALSILVLLQAGS, SEQ ID NO:48);corticotropin releasing hormone (VALLPCPPCRA, SEQ ID NO:49); betaendorphin (NAIIKNAYKKG, SEQ ID NO:50); glucagon (GSWQRSLQDTE, SEQ IDNO:51); secretin (GGSAARPAPP, SEQ ID NO:52); vasointestinal polypeptide(NALAENDTPYY, SEQ ID NO:53); neuropeptide Y (GALAEAYPSKP, SEQ ID NO:54);gonadotropin releasing hormone (GCSSQHWSYGL, SEQ ID NO:55); parathyroidhormone (VMIVMLAICFL, SEQ ID NO:56); pancreatic polypeptide(LRRYINMLTRP, SEQ ID NO:28); and calcitonin gene related peptide(LALSILVLYQA, SEQ ID NO:57) (disclosed in Grace et al., 2004, PNAS101:12836, hereby incorporated by reference in its entirety). Forexample, a chimeric peptide made from a peptide with the surface chargemotif of helix B of EPO (QEQLERALNSS, SEQ ID NO:40) joined at thecarboxy terminus to the ampipathic helix of pancreatic polypeptide(LRRYINMLTRP, SEQ ID NO:28) for a chimeric peptide. Furthermodifications may be made to the carboxy terminus of the amphipathichelix without affecting its tissue protective properties. Thus, afurther example, replacing the terminal proline of the above chimericpeptide with the sequence TR (QEQLERALNSSLRRYINMLTRTR, SEQ ID NO:41)generates a molecule with potent tissue protective activity asdemonstrated in the sciatic nerve assay (see, FIG. 1).

Additionally, instead of the above-noted helices, other tertiarystructures can be attached to the tissue protective peptides. Forexample, the helix B exterior-presenting amino acids can be linked tothe beta pleated sheet (CSLNENI, SEQ ID NO:42) found within the AB loopof EPO to form a chimeric peptide having the sequence CSLNENIQEQLERALNSS(SEQ ID NO:43), which is tissue protective (see Example 2 and Table 1).Additionally, the presenting amino acids of the terminal portion ofhelix C (ALGKA, SEQ ID NO:44, corresponding to amino acids 111, 112,113, 116, and 118 of SEQ ID NO:1) may be combined with all or part ofloop CD-partial (LGAQKEAISPPDAASAAPLRTI, SEQ ID NO:45, corresponding toamino acids 112-133 of SEQ ID NO:1). Preferably, a linking arm will bepresent between the fused peptides to provide for flexibility so thatthe joined peptides can assume the proper structural orientation to bindwith the tissue protective receptor complex. Such fusion peptides mayhave a synergistic effect, obtaining a greater tissue protective effectjointly as opposed to individually possibly through enhanced bindingwith the tissue protective receptor complex or increased biological halflife.

One of ordinary skill in the art will recognize the benefit of combiningvarious desired structural elements in to a single peptide formaximizing the tissue protective effects of such compounds. Suchchimeras may comprise amino acids peptides, and non-amino acid elements,such as linkers or bridging atoms or moieties.

5.13 Fusion Peptides

The present invention further contemplates that two or more of the abovenoted tissue protective peptides, fragment derived or chimera, may belinked to a related or unrelated protein such as erythropoietin,albumin, etc.

5.1.4 Manufacture of Tissue Protective Peptides

Tissue protective peptides of the current invention may be made usingrecombinant or synthetic techniques well known in the art. Inparticular, solid phase protein synthesis is well suited to therelatively short length of the tissue protective peptides and mayprovide greater yields with more consistent results. Additionally, thesolid phase protein synthesis may provide additional flexibilityregarding the manufacture of the tissue protective peptides. Forexample, desired chemical modifications may be incorporated into thetissue protective peptide at the synthesis stage: homocitrulline couldbe used in the synthesis of the peptide as opposed to lysine, therebyobviating the need to carbamylate the peptide following synthesis.

Synthesis

In solid-phase synthesis of a peptide an amino acid with both α-aminogroup and side chain protection is immobilized on a resin. See e.g.Nilsson, B., Soellner, M., and Raines, R. Chemical Synthesis ofProteins, Annu. Rev. Biomol. Struct. 2005.34:91-118; Meldal M. 1997.Properties of solid supports. Methods Enzymol. 289:83-104 and Songster MF, Barany G. 1997. Handles for solid-phase peptide synthesis. MethodsEnzymol. 289:126-74. Typically, two types of α-amino-protecting groupsare used: an acid-sensitive tert-butoxycarbonyl (Boc) group or abase-sensitive 9-fluorenylmethyloxycarbonyl (Fmoc) group. Wellings D A,Atherton E. 1997. Standard Fmoc protocols. Methods Enzymol. 289:44-67.After the quick and complete removal of these α-amino-protecting groupsanother protected amino acid with an activated carboxyl group can thenbe coupled to the unprotected resin-bound amine. By using an excess ofactivated soluble amino acid, the coupling reactions are forced tocompletion. The cycle of deprotection and coupling is repeated tocomplete the sequence. With side chain deprotection and cleavage, theresin yields the desired peptide. Guy CA, Fields GB. 1997.Trifluoroacetic acid cleavage and deprotection of resin-bound peptidesfollowing synthesis by Fmoc chemistry. Methods Enzymol. 289:67-83, andStewart J M. 1997. Cleavage methods following Boc-based solid-phasepeptide synthesis. Methods Enzymol.

289:29-44. Additional methods for performing solid phase proteinsynthesis are disclosed in Bang, D. & Kent, S. 2004. A One-Pot TotalSynthesis of Crambin. Angew. Chem. Int. Ed. 43:2534-2538; Bang, D.,Chopra, N., & Kent, S. 2004. Total Chemical Synthesis of Crambin. J. Am.Chem. Soc. 126:1377-1383; Dawson, P. et al. 1994. Synthesis of Proteinsby Native Chemical Ligation. Science. 266:776-779; Kochendoerfer et al.2003. Design and Chemical Synthesis of a Homogenous Polymer-ModifiedErythropoiesis Protein. Science. 299: 884-887. (Each reference recitedin this paragraph is hereby incorporated by reference in its entirety).

If necessary, smaller peptides derived from solid phase peptidesynthesis may be combined through peptide ligations such as nativechemical ligation. In this process, the thiolate of an N-terminalcysteine residue of one peptide attacks the C-terminal thioester of asecond peptide to affect transthioesterification. An amide linkage formsafter rapid S→N acyl transfer. See Dawson, P. et al. 1994. Synthesis ofProteins by Native Chemical Ligation. Science. 266:776-779, which ishereby incorporated by reference in its entirety.

Further, one of ordinary skill in the art would recognize, that thetissue protective peptides of the current invention may encompasspeptidomimetics, peptides including both naturally occurring andnon-naturally occurring amino acids, such as peptoids. Peptoids areoligomers of N-substituted glycines, glycoholic acid, thiopronine,sarcosine, and thiorphan. These structures tend to have a generalstructure of (—(C═O)—CH₂—NR—)_(n) with the R group acting as the sidechain. Such peptoids can be synthesized using solid phase synthesis inaccordance with the protocols of Simon et al., Peptoids: A molecularapproach to drug discovery, Proc. Natl. Acad. Sci. USA, 89:9367-9371(1992) and Li et al., Photolithographic Synthesis of Peptoids, J. AM.CHEM. SOC. 2004, 126, 4088-4089, each of which is hereby incorporated byreference in its entirety. Additionally, the current inventioncontemplates the use of peptidemimetics or peptide mimetics, non-peptidedrugs with properties analogous to those of the template peptide.(Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber and Friedinger (1985)TINS p. 32; and Evans et al. (1987) J. Med. Chem. 30:1229, which areincorporated by reference). Synthesis of various types ofpeptidomimetics has been reviewed for example in: Methods of OrganicChemistry (Houben-Weyl), Synthesis of Peptides andPeptidomimetics—Workbench Edition Volume E22c (Editor-in-Chief GoodmanM.) 2004 (George Theme Verlag Stuttgart, New York, hereby incorporatedby reference in its entirety).

Recombinant Techniques

A variety of host-expression vector systems may be utilized to producethe tissue protective peptides of the invention. Such host-expressionsystems represent vehicles by which the tissue protective peptide ofinterest may be produced and subsequently purified, but also representcells that may, when transformed or transfected with the appropriatenucleotide coding sequences, exhibit the modified erythropoietin geneproduct in situ. These include but are not limited to, bacteria, insect,plant, mammalian, including human host systems, such as, but not limitedto, insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus) containing the tissue protective peptidecoding sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid) containing erythropoietin-related molecule codingsequences; or mammalian cell systems, including human cell systems,e.g., HT1080, COS, CHO, BHK, 293, 3T3, harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells, e.g., metallothionein promoter, or from mammalian viruses, e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter.

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications andprocessing of protein products may be important for the function of theprotein. As known to those of ordinary skill in the art, different hostcells have specific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification andprocessing of the foreign protein expressed. To this end, eukaryotichost cells that possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells, including human hostcells, include but are not limited to HT1080, CHO, VERO, BHK, HeLa, COS,MDCK, 293, 3T3, and WI38.

For long-term, high-yield production of recombinant peptides, stableexpression is preferred. For example, cell lines that stably express therecombinant tissue protective cytokine-related molecule gene product maybe engineered. Rather than using expression vectors that contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements, e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,and the like, and a selectable marker. Following the introduction of theforeign DNA, engineered cells may be allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci that in turn can be cloned andexpanded into cell lines. This method may advantageously be used toengineer cell lines that express the tissue-protective product. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that affect the endogenous activity of theEPO-related molecule gene product.

Further Modifications

Additional modifications can be made to the tissue protective peptides.For example, the peptide may be synthesized with one or more (D)-aminoacids. The choice of including an (L)- or (D)-amino acid into a peptideof the present invention depends, in part, upon the desiredcharacteristics of the peptide. For example, the incorporation of one ormore (D)-amino acids can confer increasing stability on the peptide invitro or in vivo. The incorporation of one or more (D)-amino acids canalso increase or decrease the binding activity of the peptide asdetermined, for example, using the bioassays described herein, or othermethods well known in the art.

Replacement of all or part of a sequence of (L)-amino acids by therespective sequence of entatiomeric (D)-amino acids renders an opticallyisomeric structure in the respective part of the polypeptide chain.Inversion of the sequence of all or part of a sequence of (L)-aminoacids renders retro-analogues of the peptide. Combination of theenantiomeric (L to D, or D to L) replacement and inversion of thesequence renders retro-inverso-analogues of the peptide. It is known tothose skilled in the art that enantiomeric peptides, theirretro-analogues, and their retro-inverso analogues maintain significanttopological relationship to the parent peptide, and especially highdegree of resemblance is often obtained for the parent and itsretro-inverso-analogues. This relationship and resemblance can bereflected in biochemical properties of the peptides, especially highdegrees of binding of the respective peptides and analogs to a receptorprotein. The synthesis of the properties of retro-inverso anologues ofpeptides have been discussed for example in Methods of Organic Chemistry(Houben-Weyl), Synthesis of Peptides and Peptidomimetics—WorkbenchEdition Volume E22c (Editor-in-chief Goodman M.) 2004 (George ThiemeVerlag Stuttgart, New York), and in references cited therein, all ofwhich are hereby incorporated by reference herein in their entireties.

Amino acid “modification” refers to the alteration of a naturallyoccurring amino acid to produce a non-naturally occurring amino acid.Derivatives of the peptides of the present invention with non-naturallyoccurring amino acids can be created by chemical synthesis or by sitespecific incorporation of unnatural amino acids into polypeptides duringbiosynthesis, as described in Christopher J. Noren, Spencer J.Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, 1989 Science,244:182-188, hereby incorporated by reference herein in its entirety.

Peptide mimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent therapeutic orprophylactic effect. Generally, peptidomimetics are structurally similarto a paradigm polypeptide (i.e., a polypeptide that has a biochemicalproperty or pharmacological activity), but have one or more peptidelinkages optionally replaced by a linkage selected from the groupconsisting of: —CH₂—NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH—(cis and trans),—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methods known in the art andfurther described in the following references: Spatola, A. F. in“Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins,” B.Weinstein, eds., Marcel Dekker, New York, p 267 (1983); Spatola, A. F.,Vega Data (March 1983), Vol. 1. Issue 3, “Peptide BackboneModifications” (general review); Morely, J. S., Trends Pharma Sci (1980)pp. 463-468 (general review); Hudson, D. et al., (1979) Int J Pept ProtRe 14: 177-185 (—CH—NH—, —CH₂—CH₂—); Spatola, A. F. et al., (1986) LifeSci 38:1243-1249 (—CH₂—S); Hann, M. M., (1982) J Chem Soc Perkin Trans1307-314 (—CH═CH—, cis and trans); Almquist, R. G. et al., (1980) J MedChem 23: 1392 (—COCH₂—); Jennings-White, C et al., (1982) TetrahedronLett 23:2533 (—COCH₂—); Szelke, M et al., European Appln. EP 45665(1982) CA: 97: 39405 (1982) (—CH(OH)CH₂—); Holladay, M. W. et al.,(1983) Tetrahedron Lett 24:4401-4404 (—C(OH)CH₂—); and Hruby, V. J.,(1982) Life Sci 31:189-199 (—CH₂—S—); each of which is incorporatedherein by reference.

In another embodiment, a particularly preferred non-peptide linkage is—CH₂NH—. Such peptide mimetics may have significant advantages overpolypeptide embodiments, including, for example: more economicalproduction, greater chemical stability, enhanced pharmacologicalproperties (half-life, absorption, potency, efficacy, etc.), alteredspecificity (e.g., a broad-spectrum of biological activities), reducedantigenicity, and others.

A variety of designs for peptide mimetics are possible. For example,cyclic peptides, in which the necessary conformation is stabilized bynon-peptides, are specifically contemplated, U.S. Pat. No. 5,192,746 toLobl, et al., U.S. Pat. No. 5,576,423 to Aversa, et al., U.S. Pat. No.5,051,448 to Shashoua, and U.S. Pat. No. 5,559,103 to Gaeta, et al., allhereby incorporated by reference, describe multiple methods for creatingsuch compounds. Synthesis of nonpeptide compounds that mimic peptidesequences is also known in the art. Eldred et al., J. Med. Chem. 37:3882(1994), hereby incorporated by reference herein in its entirety)describe non-peptide antagonists that mimic the peptide sequence.Likewise. Ku et al., J. Med. Chem. 38:9 (1995) (hereby incorporated byreference herein in its entirety) further elucidates the synthesis of aseries of such compounds.

Further modifications following synthesis may be implemented. Forexample, the tissue protective peptides may be further chemicallymodified, i.e. carbamylated, acetylated, succinylated, etc., inaccordance with U.S. patent application Ser. No. 10/188,905, whichpublished as 20030072737-A1 on Apr. 17, 2003 and discloses chemicallymodified EPO, and in accordance with U.S. patent application Ser. No.10/612,665, filed Jul. 1, 2003, and U.S. patent application Ser. No.09/753,132, filed Dec. 29, 2000, which are incorporated by referenceherein in their entirety.

Additionally, the tissue protective peptides may consist of recombinanttissue protective peptides—muteins. The disclosed mutations may includesubstitutions, deletions, including internal deletions, additions,including additions yielding fusion proteins, or conservativesubstitutions of amino acid residues within and/or adjacent to the aminoacid sequence, but that result in a “silent” change, andnon-conservative amino acid changes and larger insertions and deletions,as previously disclosed in PCT/US03/20964 entitled Recombinant TissueProtective Cytokines and Encoding Nucleic Acids Thereof for Protection,Restoration, and Enhancement of Responsive Cells, Tissues, and Organs(which is incorporated by reference herein in its entirety)

Either conservative or non-conservative amino acid substitutions can bemade at one or more amino acid residues. Both conservative andnon-conservative substitutions can be made. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Genetically encoded amino acids can bedivided into four families: (1) acidic=aspartate, glutamate; (2)basic=lysine, arginine, histidine; (3) nonpolar (hydrophobic)=cysteine,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, glycine, tyrosine; and (4) unchargedpolar=asparagine, glutamine, serine, threonine. Non-polar may besubdivided into: strongly hydrophobic=alanine, valine, leucine,isoleucine, methionine, phenylalanine and moderatelyhydrophobic=glycine, proline, cysteine, tyrosine, tryptophan. Inalternative fashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (See, for example, Biochemistry, 4th ed., Ed. by L. Stryer,W H Freeman and Co., 1995, which is incorporated by reference herein inits entirety).

Alternatively, mutations can be introduced randomly along all or part ofthe coding sequence of a tissue protective peptide, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded peptide can be expressed recombinantly and theactivity of the recombinant tissue protective peptide can be determined.

In another embodiment, the tissue protective peptide may be furthermodified through the additions of polymers (such as polyethyleneglycol), sugars, or additional proteins (such as a fusion construct) inan effort to extend the half-life of the tissue protective peptide orenhance the peptide's tissue protective effects. Examples of suchmodifications are disclosed within WO/04022577 A3 and WO/05025606 A1,which are incorporated herein by reference.

5.2 Assays for Testing Tissue Protective Peptides 5.2.1 BiologicalScreens or Assays

Tissue protective peptides in accordance with the present invention maybe tested for tissue protective activity, e.g., protecting cells,tissues or organs. Protective activities may be further tested using invitro and in vivo assays. In vitro tests that are indicative of tissueprotective activity include, for example, cell proliferation assays,cell differentiation assays, or detecting the presence of proteins ornucleic acids upregulated by tissue protective receptor complex, e.g.tissue protective cytokine receptor complex, activity, e.g., nucleolin,neuroglobin, cytoglobin, or frataxin. Neuroglobin, for example, may beinvolved in facilitating the transport or the short-term storage ofoxygen. Therefore, oxygen transport or storage assays may be used as anassay to identify or screen for compounds which modulate tissueprotective activity.

Neuroglobin is expressed in cells and tissues of the central nervoussystem in response to hypoxia or ischemia and may provide protectionfrom injury (Sun et al. 2001, PNAS 98:15306-15311; Schmid et al., 2003,J. Biol. Chem. 276:1932-1935, each of which is incorporated by referenceherein in its entirety). Cytoglobin may play a similar role inprotection, but is expressed in a variety of tissues at varying levels(Pesce et al., 2002, EMBO 3:1146-1151, which is incorporated byreference herein in its entirety). In one embodiment of the invention,the levels of an upregulated protein in a cell may be measured beforeand after contacting the tissue protective peptide to a cell. In certainembodiments, the presence of an upregulated protein associated withtissue protective activity in a cell, may be used to confirm the tissueprotective activities of a peptide.

Nucleolin may protect cells from damage. It plays numerous roles incells including modulation of transcription processes, sequence specificRNA-binding protein, cytokinesis, nucleogensis, signal transduction,apoptosis induced by T-cells, chromatin remodelling, or replication. Itcan also function as a cell surface receptor DNA/RNA helicase,DNA-dependent ATPase, protein shuttle, transcription factor component,or transcriptional repressor (Srivastava and Pollard, 1999, FASEB J.,13:1911-1922; and Ginisty et al., 1999, J. Cell Sci., 112:761-772, eachof which is incorporated by reference herein in its entirety).

Frataxin is a protein involved with mitochondrial iron metabolism andhas previously been shown to be strongly up-regulated by EPO both invivo and in vitro (Sturm et al. (2005) Eur J Clin Invest 35: 711, whichis incorporated by reference herein in its entirety)

Expression of an upregulated protein may be detected by detecting mRNAlevels corresponding to the protein in a cell. The mRNA can behybridized to a probe that specifically binds a nucleic acid encodingthe upregulated protein. Hybridization may consist of, for example,Northern blot, Southern blot, array hybridization, affinitychromatography, or in situ hybridization.

Tissue protective activity of the polypeptide of the invention can alsobe detected using in vitro neuroprotection assays. For example, primaryneuronal cultures may be prepared from new born rat hippocampi bytrypsinization, and cultured as by any method known in the art and/ordescribed herein e.g. in MEM-II growth medium (Invitrogen), 20 mMD-glucose, 2 mM L-glutamine, 10% Nu-serum (bovine; Becton Dickinson,Franklin Lakes, N.J.), 2% B27 supplement (Invitrogen), 26.2 mM NaHCO₃,100 U/ml penicillin, and 1 mg/ml streptavidin (see, e.g., Leist et al.,2004, Science 305:239-242, hereby incorporated by reference in itsentirety). One day after seeding, 1 μM cytosinearabino-furanoside isadded. Thirteen day old cultures are then preincubated with increasingdoses of EPO or CEPO (3-3000 μM) for 24 h. On day 14, the medium isremoved and the cultures challenged with 300 μM NMDA in PBS at RT. After5 min, pre-conditioned medium is returned to the cultures which are thenreturned to the incubator for 24 h. The cells are fixed inparaformaldehyde, stained by Hoechst 33342 (Molecular Probes, Eugene,Oreg.) and condensed apoptotic nuclei may be counted. NGF (50 ng/ml) andMK801 (1 μM) are included as positive controls.

Animal model systems can be used to demonstrate the tissue protectiveactivity of a compound or to demonstrate the safety and efficacy of thecompounds identified by the screening methods of the invention describedabove. The compounds identified in the assays can then be tested forbiological activity using animal models for a type of tissue damage,disease, condition, or syndrome of interest. These include animalsengineered to contain the tissue protective receptor complex coupled toa functional readout system, such as a transgenic mouse.

Animal models that can be used to test the efficacy of the cell ortissue protective activity of an identified compound include, forexample, protection against the onset of acute experimental allergicencephalomyelitis (EAE; see, Example 12) in Lewis rats, restoration orprotection from diminished cognitive function in mice after receivingbrain trauma, cerebral ischemia (“stroke”; Example 5) or seizuresstimulated by excitotoxins (Brines et al., 2000, PNAS, 97:10295-10672,which is incorporated by reference herein in its entirety), protectionfrom induced retinal ischemia (Rosenbaum et al., 1997, Vis. Res.37:3443-51 which is incorporated by reference herein in its entirety),protection from injury to the sciatic nerve (see, Example 2), andprotection from ischemia-reperfusion injury to the heart (in vitrocardiomyocyte studies and in vivo ischemia-reperfusion injury, see,e.g., Calvillo et al., 2003, PNAS 100:4802-4806 and Fiordaliso et al.,2005, PNAS 102:2046-2051, each of which is hereby incorporated byreference in its entirety). Such assays are described in further detailin Grasso et al. (2004) Med Sci Monit 10: BR1-3 or PCT publication no.WO02/053580, each of which is incorporated by reference herein in itsentirety. The in vivo methods described therein are directed towardsadministration of EPO, however, tissue protective proteins administeredin place of EPO have been identified to also exhibit similar biologicactivity, e.g., Leist et al. (2004) Science 305: 239-242, which isincorporated by reference herein in its entirety. Peptides may besubstituted for testing as well. Other assays for determining tissueprotective activity of a peptide are well known to those of skill in theart.

5.2.2 Cell Binding Assays

Alternatively, cell binding assays can be for evaluation of thepolypeptides of the invention. For example, the tissue protectivepeptide of interest can be bound to a biological marker such as afluorescent or radiolabled marker for ease of detection and tested forbinding to transfected BaF3 cells expressing EPOR and/or β_(c) receptor.In a 96 well plate, eight 1:2 serial dilutions of the tissue protectivepeptide of interest in growth medium (RPMI 1640, 10% fetal bovine serum,1 mM sodium pyruvate, 2 mM L-glutamine) are plated, such that the finalvolume in each well is about 100 μl. The BaF3 parental line and BaF3cells transfected with EPOR and/or β_(c) receptor can be washed threetimes in growth media (see above), pellets resuspended in growth medium,and cells counted and diluted in growth media to 5,000 cells/100 μl. 100μl of diluted cells are then added to each peptide dilution. The assayplate is then incubated in a 37° C. incubator for three to four days.The plate/cells are then washed and the plate is read on a fluorescentplate reader or by other suitable method to detect the level ofbiomarker associated with the biological activity of the tissueprotective peptide of interest.

Similarly, a competitive assay can be utilized to determine if a tissueprotective peptide is tissue protective. In the competitive assay, acompound known to be tissue protective including, but not limited to,tissue protective cytokines such as those disclosed in U.S. patentapplication Ser. Nos. 10/188,905 and 10/185,841 (each of which isincorporated by reference herein in its entirety), can be attached to asuitable bio marker.

In a 96 well plate eight 1:2 serial dilutions of a known tissueprotective compound/biomarker in suitable growth medium, and the samedilution series of the known tissue protective compound/biomarker and anexcess of the tissue protective peptide of interest are plated. Thefinal volume of each dilution should be about 100 μl. Once again, theBaF3 cells are seeded into the plates as disclosed supra and allowed toincubate. After an appropriate amount of time, the cells are washed andthe plate is read on a fluorescent plate reader or by any other suitablemethod known in the art to detect the biomarker. If the readout of theplates and/or wells containing the known tissue protectivecompound/biomarker and tissue protective peptide of interest is lessthan the readout of the plates containing only the known tissueprotective compound/biomarker then the tissue protective peptide ofinterest is tissue protective.

5.2.3 Cytokine and Cell Proliferation/Differentiation Activity

Many protein factors discovered to date, including all known cytokines,have exhibited activity in one or more factor-dependent cellproliferation assays, and hence these assays serve as a convenientconfirmation of cytokine activity. The activity of a tissue protectivepeptide can be evidenced by any one of a number of routine factordependent cell proliferation assays for cell lines including, withoutlimitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+),2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK. These cellsare cultured in the presence or absence of a tissue protective peptide,and cell proliferation is detected by, for example, measuringincorporation of tritiated thymidine or by colorimetric assay based onthe metabolic breakdown of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Mosman, 1983, J. Immunol. Meth. 65:55-63,which is incorporated by reference herein in its entirety).

5.2.4 Other Assays

If a tissue protective peptide exhibits a tissue protective activity,one of ordinary skill in the art would recognize that it would bebeneficial to verify the result using one of the neuroprotective andtissue protective assays known to those skilled in the art, such as, butnot limited to, P-19 and PC-12 cell assays. Additionally, various invivo models such as animal models related to spinal cord injury,ischemic stroke, peripheral nerve damage, heart, eyes, kidneys, etc.would be helpful in further characterizing the tissue protectivepeptide. Suitable in vitro and in vivo assays are disclosed in U.S.patent application Ser. Nos. 10/188,905 and 10/185,841, each of which isincorporated by reference herein in its entirety.

5.3 Therapeutic Use

One of ordinary skill in the art would recognize that the tissueprotective peptides of the current invention are useful as therapeuticsfor treatment or prevention of various diseases, disorders, andconditions. One skilled in the art would also recognize that suchpeptides can be used to achieve modulation of a tissue protectivereceptor complex, e.g., tissue protective cytokine complex. Both invitro and in vivo techniques that can be used for assessing thetherapeutic indications of, for example, the compounds identified by theinventive assays disclosed above are disclosed in PCT Application No.PCT/US01/49479, U.S. patent application Ser. Nos. 10/188,905 and10/185,841, incorporated herein by reference.

The aforementioned tissue protective peptides of the invention may beuseful generally for the prevention, therapeutic treatment, orprophylactic treatment of human diseases or disorders of the centralnervous system or peripheral nervous system which have primarilyneurological or psychiatric symptoms, ophthalmic diseases,cardiovascular diseases, cardiopulmonary diseases, respiratory diseases,kidney, urinary and reproductive diseases, bone diseases, skin diseases,connective tissue diseases, gastrointestinal diseases and endocrine andmetabolic abnormalities. Examples of use include, but are not limitedto, protection against and repair of injury resulting from trauma andresulting inflammation to the brain (ischemic stroke, blunt trauma,subarachnoid hemorrhage), spinal cord (ischemia, blunt force trauma),peripheral nerves (sciatic nerve injury, diabetic neuropathy, carpaltunnel syndrome), retinal (macular edema, diabetic retinopathy,glaucoma), and heart (myocardial infarct, chronic heart failure). Inparticular, such diseases, disorders, and conditions include hypoxicconditions, which adversely affect responsive tissues, such as excitabletissues in the central nervous system tissue, peripheral nervous systemtissue, or cardiac tissue or retinal tissue such as, for example, brain,heart, or retina/eye. Therefore, the tissue protective peptides of theinvention can be used to treat or prevent damage to responsive tissueresulting from hypoxic conditions in a variety of conditions andcircumstances. Non-limiting examples of such conditions andcircumstances are provided in the table herein below.

The tissue protective polypeptides are also of interest in themodulation of stem cell activity. It has been established that cytokinesexhibiting tissue protective activity, e.g. EPO, are able to mobilizestem cells, stimulating the migration to regions of injury and aidingthe repair process, e.g. in a regenerative role. For example, inexperimental stroke, EPO mediates the migration of neuroblasts into aregion of ischemic injury to regenerate neurons during the period ofrecovery (Tsai et al, J. Neurosci (2006) 26:1269-74, incorporated hereinby reference in its entirety). As another example, EPO and CEPO mobilizeendothelial progenitor cells from the bone marrow into the circulation.These cells then home to distance regions and are involved in theformation of new blood vessels (for effect of EPO, see, Bahlmann et al,2003, Kidney Int. 64:1648-1652, incorporated by reference herein in itsentirety). While not wishing to be bound to any particular theory, theisolated polypeptides disclosed herein are believed to have a similareffect on the migration of stem cells.

In the example of the protection of neuronal tissue pathologiestreatable and preventable using tissue protective peptides of theinvention, such pathologies include those which result from reducedoxygenation of neuronal tissues. Any condition which reduces theavailability of oxygen to neuronal tissue, resulting in stress, damage,and finally, neuronal cell death, can be treated using tissue protectivepeptides of the present invention. Generally referred to as hypoxiaand/or ischemia, these conditions arise from or include, but are notlimited to, stroke, vascular occlusion, prenatal or postnatal oxygendeprivation, suffocation, choking, near drowning, carbon monoxidepoisoning, smoke inhalation, trauma, including surgery and radiotherapy,asphyxia, epilepsy, hypoglycemia, chronic obstructive pulmonary disease,emphysema, adult respiratory distress syndrome, hypotensive shock,septic shock, anaphylactic shock, insulin shock, sickle cell crisis,cardiac arrest, dysrhythmia, nitrogen narcosis, and neurologicaldeficits caused by heart-lung bypass procedures.

In one embodiment, for example, the tissue protective peptides of thepresent invention identified using the inventive assay could beadministered alone or as part of a composition to prevent injury ortissue damage resulting from risk of injury or tissue damage prior to,during, or subsequent to a surgical procedure or a medical procedure.For example, surgical procedures may include tumor resection or aneurysmrepair and medical procedures may include labor or delivery. Otherpathologies caused by or resulting from hypoglycemia which are treatableusing tissue protective peptides of the present invention includeinsulin overdose, also referred to as iatrogenic hyperinsulinemia,insulinoma, growth hormone deficiency, hypocortisolism, drug overdose,and certain tumors.

Other pathologies resulting from excitable neuronal tissue damageinclude seizure disorders, such as epilepsy, convulsions, or chronicseizure disorders. Other treatable conditions and diseases include, butare not limited to, diseases such as stroke, multiple sclerosis,hypotension, cardiac arrest, Alzheimer's disease, Parkinson's disease,cerebral palsy, brain or spinal cord trauma, AIDS dementia, age-relatedloss of cognitive function, memory loss, amyotrophic lateral sclerosis,seizure disorders, alcoholism, retinal ischemia, optic nerve damageresulting from glaucoma, and neuronal loss.

The specific tissue protective peptides of the present invention may beused to treat or prevent inflammation resulting from disease conditionsor various traumas, such as physically or chemically inducedinflammation. The tissue protective peptides are also contemplated forthe treatment and prevention of inflammatory conditions in one or moreorgans or tissues including, but not limited to, the brain, spinal cord,connective tissue, heart, lung, kidney and urinary tract, pancreas, eyesand prostate. Non-limiting examples of such trauma include tendonitis,angitis, chronic bronchitis, pancreatitis, osteomyelitis, rheumatoidarthritis, glomerulonephritis, optic neuritis, temporal arteritis,encephalitis, meningitis, transverse myelitis, dermatomyositis,polymyositis, necrotizing fascilitis, hepatitis, and necrotizingenterocolitis. Further the tissue protective cytokines may used to treator prevent inflammation resulting from ischemic and non-ischemicconditions including, but not limited to, allergies, rheumatic diseases,sports related injuries, infections including viral, fungal, andbacterial. The inflammation may be acute or chronic. Furtherapplications in the field of inflammation are noted withinPCT/US2004/031789 filed Sep. 29, 2004 and published as WO 2005/032467,hereby incorporated by reference in its entirety.

The specific tissue protective peptides of the present invention may beused to treat central nervous and peripheral nervous system diseasesresulting from demyelination or impairment of the myelin sheath. Thesediseases are defined as mainly involving inflammatory myelin sheathlesions of unknown origin, with the exception of myelination deficiencydiseases, such as leukodystrophy, and diseases due to obvious causes.Multiple sclerosis (MS) is a typical disease among demyelinatingdiseases, and pathologically, it is characterized by changes, mainly,inflammatory demyelination, and gliosis. Since its etiology is unknown,its diagnosis is made based on its clinical features, i.e., spatialmultiplicity and multiplicity over time of central nervous systemlesions. Furthermore, acute disseminated encephalomyelitis (ADEM),inflammatory diffuse sclerosis, acute and subacute necrotizinghemorrhagic encephalomyelitis, and transverse myelitis are included indemyelinating diseases. Also, peripheral nervous tissues rely uponSchwann's cells to maintain the myelin sheath, if these cells areimpaired, peripheral demyelinating disease is caused.

The tissue protective peptides of the present invention may be used totreat or prevent conditions of, and damage to the heart including anychronic or acute pathological event involving the heart and/orassociated tissue (e.g., the pericardium, aorta and other associatedblood vessels), including ischemia-reperfusion injury; congestive heartfailure; cardiac arrest; myocardial infarction; atherosclerosis, mitralvalve leakage, atrial flutter, cardiotoxicity caused by compounds suchas drugs (e.g., doxorubicin, herceptin, thioridazine and cisapride);cardiac damage due to parasitic infection (bacteria, fungi, rickettsiae,and viruses, e.g., syphilis, chronic Trypanosoma cruzi infection);fulminant cardiac amyloidosis; heart surgery; heart transplantation;angioplasty, laparoscopic surgery, traumatic cardiac injury (e.g.,penetrating or blunt cardiac injury, and aortic valve rupture), surgicalrepair of a thoracic aortic aneurysm; a suprarenal aortic aneurysm;cardiogenic shock due to myocardial infarction or cardiac failure;neurogenic shock and anaphylaxis. The tissue protective peptides of thecurrent invention may also be used to treat those individuals at riskfor heart disease such as cardiac failure (i.e., where the heart is notable to pump blood at a rate required by the metabolizing tissues, orwhen the heart can do so only with an elevated filling pressure). Suchat risk patients would include patients having or being at risk ofhaving cardiac infarction, coronary artery disease, myocarditis,chemotherapy, cardiomyopathy, hypertension, valvular heart diseases(most often mitral insufficiency and aortic stenosis) and toxin-inducedcardiomyopathy (e.g. ethanol, cocaine, etc.) and the like.

The tissue protective peptides of the present invention may be used totreat or prevent conditions of, and damage to, the eyes, e.g., retinaltissue. Such disorders include, but are not limited to retinal ischemia,macular degeneration, retinal detachment, retinitis pigmentosa,arteriosclerotic retinopathy, hypertensive retinopathy, retinal arteryblockage, retinal vein blockage, hypotension, and diabetic retinopathy.

In another embodiment, the tissue protective peptides of the presentinvention and principles of the invention may be used to prevent ortreat injury resulting from radiation damage to responsive tissue. Afurther utility of the tissue protective peptides of the presentinvention is in the treatment of poisoning, such as neurotoxin poisoning(e.g., domoic acid shellfish poisoning), toxins (ethanol, cocaine,etc.), as the result of chemotherapeutic agents of radiation exposure;neurolathyrism; Guam disease; amyotrophic lateral sclerosis; andParkinson's disease.

As mentioned above, the present invention is also directed to tissueprotective peptides of the present invention for use in enhancing tissuefunction in responsive cells, tissues and organs in a mammal byperipheral administration of a tissue protective cytokine as describedabove. Various diseases and conditions are amenable to treatment usingthis method. For example this method is useful for enhancing function inexcitable tissues resulting in an increase in cognitive function even inthe absence of any condition or disease. Further, the tissue protectivecytokines are useful for improving the quality of wound healing,reducing the time required to heal, improving the quality of the healedtissues and reducing the incidence of adhesions resulting from thewound. See PCT/US2004/031789 filed Sep. 29, 2004 and published as WO2005/032467, hereby incorporated by reference in its entirety. Theseuses of the present invention are describe in further detail below andinclude enhancement of learning and training in both human and non-humanmammals.

Conditions and diseases treatable or preventable using tissue protectivepeptides of the present invention directed to the central nervous systeminclude but are not limited to mood disorders, anxiety disorders,depression, autism, attention deficit hyperactivity disorder, andcognitive dysfunction. These conditions benefit from enhancement ofneuronal function. Other disorders treatable in accordance with theteachings of the present invention include sleep disruption, forexample, sleep apnea and travel-related disorders; subarachnoid andaneurismal bleeds, hypotensive shock, concussive injury, septic shock,anaphylactic shock, and sequelae of various encephalitides andmeningitides, for example, connective tissue disease-relatedcerebritides such as lupus. Other uses include prevention of orprotection from poisoning by neurotoxins, such as domoic acid shellfishpoisoning, neurolathyrism, and Guam disease, amyotrophic lateralsclerosis, Parkinson's disease; postoperative treatment for embolic orischemic injury; whole brain irradiation; sickle cell crisis; andeclampsia.

A further group of conditions treatable or preventable using tissueprotective peptides of the present invention include mitochondrialdysfunction, of either a hereditary or acquired nature, which are thecause of a variety of neurological diseases typified by neuronal injuryand death. For example, Leigh disease (subacute necrotizingencephalopathy) is characterized by progressive visual loss andencephalopathy, due to neuronal drop out, and myopathy. In these cases,defective mitochondrial metabolism fails to supply enough high energysubstrates to fuel the metabolism of excitable cells. A tissueprotective peptide optimizes failing function in a variety ofmitochondrial diseases. As mentioned above, hypoxic conditions adverselyaffect excitable tissues. The excitable tissues include, but are notlimited to, central nervous system tissue, peripheral nervous systemtissue, and heart tissue. In addition to the conditions described above,the tissue protective peptides of the present invention are useful inthe treatment of inhalation poisoning such as carbon monoxide and smokeinhalation, severe asthma, adult respiratory distress syndrome, andchoking and near drowning. Further conditions which create hypoxicconditions or by other means induce responsive tissue, such as excitabletissue damage include hypoglycemia that may occur in inappropriatedosing of insulin, or with insulin-producing neoplasms (insulinoma).

Various neuropsychologic disorders which are described to originate fromexcitable tissue damage are treatable using tissue protective peptidesof the present invention. Chronic disorders in which neuronal damage isinvolved and for which treatment or preventable by the present inventionis provided include disorders relating to the central nervous systemand/or peripheral nervous system including age-related loss of cognitivefunction and senile dementia, chronic seizure disorders, Alzheimer'sdisease, Parkinson's disease, dementia, memory loss, amyotrophic lateralsclerosis, multiple sclerosis, tuberous sclerosis, Wilson's Disease,cerebral and progressive supranuclear palsy, Guam disease, Lewy bodydementia, prion diseases, such as spongiform encephalopathies, e.g.,Creutzfeldt-Jakob disease, Huntington's disease, myotonic dystrophy,Freidrich's ataxia and other ataxias, as well as Gilles de la Tourette'ssyndrome, seizure disorders such as epilepsy and chronic seizuredisorder, stroke, brain or spinal cord trauma, AIDS dementia,alcoholism, autism, retinal ischemia, glaucoma, autonomic functiondisorders such as hypertension and sleep disorders, and neuropsychiatricdisorders that include, but are not limited to schizophrenia,schizoaffective disorder, attention deficit disorder, dysthymicdisorder, major depressive disorder, mania, obsessive-compulsivedisorder, psychoactive substance use disorders, anxiety, panic disorder,as well as unipolar and bipolar affective disorders. Additionalneuropsychiatric and neurodegenerative disorders include, for example,those listed in the American Psychiatric Association's Diagnostic andStatistical Manual of Mental Disorders (DSM), the most current versionof which in incorporated herein by reference in its entirety.

A further group of conditions treatable or preventable using tissueprotective peptides of the present invention include kidney diseasessuch as renal failure, acute and chronic. Blood supply to the kidneyscan be cut off due to several causes including shock from infectionsinvading the bloodstream (septicemia), internal or externalhemorrhaging, loss of fluid from the body as a result of severe diarrheaor burns, reactions to transfusions, cardiac arrest or arythmias,surgical trauma and kidney transplantations. The reduced flow of bloodto the kidneys resulting from the above conditions may reduced bloodflow to dangerously low levels for a time period great enough to causethe development of acute renal failure. The depressed blood flow alsoresults in necrosis, or tissue death, in the kidney, damaging the renaltubular cells. Renal failure may also result from diseases (interstitialand diabetic) nephrotic syndromes, infections, injury (CPB-induced),toxins (contrast-induced, chemotherapy-induced, cyclosporine),autoimmune inflammation (e.g. Lupus, erythrotosis, etc.) The tissueprotective peptides of the current invention assist in the repair orprevention of this damage helping to ameliorate acute renal failure.

The following table lists additional exemplary, non-limiting indicationsas to the various conditions and diseases amenable to treatment by theaforementioned tissue protective peptides.

Cell, tissue or Dysfunction or organ pathology Condition or disease TypeHeart Ischemia Coronary artery disease Acute, chronic Stable, unstableMyocardial infarction Dressler's syndrome Angina Congenital heartdisease Valvular Cardiomyopathy Prinzmetal angina Cardiac ruptureAneurysmatic Septal perforation Angiitis Arrhythmia Tachy-,bradyarrhythmia Stable, unstable Supraventricular, Hypersensitivecarotid sinus ventricular node Conduction abnormalities Congestive heartfailure Left, right, bi-ventricular, Cardiomyopathies, such as systolic,diastolic idiopathic familial, infective, metabolic, storage disease,deficiencies, connective tissue disorder, infiltration and granulomas,neurovascular Myocarditis Autoimmune, infective, idiopathic Corpulmonale Radiation injury Blunt and penetrating trauma Toxins Cocainetoxicity, adriamycin Vascular Hypertension Primary, secondaryDecompression sickness Fibromuscular hyperplasia Aneurysm Dissecting,ruptured, enlarging Lungs Obstructive Asthma Chronic bronchitis,Emphysema and airway obstruction Ischemic lung disease Pulmonaryembolism, Pulmonary thrombosis, Fat embolism Environmental lung diseasesIschemic lung disease Pulmonary embolism Pulmonary thrombosisInterstitial lung disease Idiopathic pulmonary fibrosis CongenitalCystic fibrosis Cor pulmonale Trauma Pneumonia and Infectious,parasitic, pneumonitides toxic, traumatic, burn, aspiration SarcoidosisPancreas Endocrine Diabetes mellitus, type I Beta cell failure,dysfunction and II Diabetic neuropathy Other endocrine cell failure ofthe pancreas Exocrine Exocrine pancreas failure pancreatitis BoneOsteopenia Primary Hypogonadism Secondary immobilisation PostmenopausalAge-related Hyperparathyroidism Hyperthyroidism Calcium, magnesium,phosphorus and/or vitamin D deficiency Osteomyelitis Avascular necrosisTrauma Paget's disease Skin Alopecia Areata Primary Totalis SecondaryMale pattern baldness Vitiligo Localized Primary Generalized secondaryUlceration Diabetic Pressure sores, pressure ulcers, Decubitis bed soresPeripheral vascular disease Surgical wounds, lacerations Burn injuriesAutoimmune Lupus erythematosus, disorders Sjogren's syndrome, Rheumatoidarthritis, Glomerulonephritis, Angiitis Langerhan's histiocytosis EyeOptic neuritis Blunt and penetrating injuries, Infections, Sarcoid,Sickle C disease, Retinal detachment, Temporal arteritis Retinalischemia, Macular degeneration, Retinitis pigmentosa, Arterioscleroticretinopathy, Hypertensive retinopathy, Retinal artery blockage, Retinalvein blockage, Hypotension, Diabetic retinopathy, glaucoma and Macularedema Embryonic and Asphyxia fetal disorders Ischemia CNS Chronicfatigue syndrome, acute and chronic hypoosmolar and hyperosmolarsyndromes, AIDS Dementia, Electrocution Cerebral malaria EncephalitisRabies, Herpes, Meningitis Subdural hematoma Nicotine addiction Drugabuse and Cocaine, heroin, crack, withdrawal marijuana, LSD, PCP,poly-drug abuse, ecstasy, opioids, sedative hypnotics, amphetamines,caffeine Obsessive-compulsive disorders Spinal stenosis, Transversemyelitis, Guillian Barre, Trauma, Nerve root compression, Tumoralcompression, Heat stroke ENT Tinnitus Meuniere's syndrome Hearing lossTraumatic injury, barotraumas Kidney Renal failure Acute, chronicVascular/ischemic, interstitial disease, diabetic kidney disease,nephrotic syndromes, infections, injury, contrast-induced,chemotherapy-induced, cyclosporine, CPB-induced, or preventive Radiationinjury Henoch Schonlein purpura Striated muscle Autoimmune disordersMyasthenia gravis Dermatomyositis Polymyositis Myopathies Inheritedmetabolic, endocrine and toxic Heat stroke Crush injury RhabdomyolysisMitochondrial disease Infection Necrotizing fasciitis Sexual Central andperipheral Impotence secondary to dysfunction (e.g. erectiledysfunction) medication, (diabetes) Liver Hepatitis Viral, bacterial,parasitic Ischemic disease Cirrhosis, fatty liver Infiltrative/metabolicdiseases Gastrointestinal Ischemic bowel disease Inflammatory boweldisease Necrotizing enterocolitis Organ Treatment of donor andtransplantation recipient Reproductive Infertility Vascular tractAutoimmune Uterine abnormalities Implantation disorders EndocrineGlandular hyper- and hypofunction General Shock Septic, hemodynamicParasitemia Malaria, trypanosomiasis, Leshmaniasis

As mentioned above, these diseases, disorders or conditions are merelyillustrative of the range of benefits provided by the tissue protectivepeptides of the present invention. Accordingly, this invention generallyprovides preventative, therapeutic, or prophylactic treatment of theconsequences of mechanical trauma or of human diseases. Prevention ortherapeutic or prophylactic treatment for diseases, disorders orconditions of the CNS and/or peripheral nervous system are contemplated.Prevention or therapeutic or prophylactic treatment for diseases,disorders or conditions which have a psychiatric component is provided.Prevention or therapeutic or prophylactic treatment for diseases,disorders or conditions including but not limited to those having anophthalmic, cardiovascular, cardiopulmonary, respiratory, kidney,urinary, reproductive, gastrointestinal, endocrine, or metaboliccomponent is provided.

In one embodiment, such a pharmaceutical composition comprising a tissueprotective peptide can be administered systemically to protect orenhance the target cells, tissue or organ. Such administration may beparenterally, via inhalation, or transmucosally, e.g., orally, nasally,rectally, intravaginally, sublingually, ocularly, submucosally ortransdermally. Preferably, administration is parenteral, e.g., viaintravenous or intraperitoneal injection, and also including, but is notlimited to, intra-arterial, intramuscular, intradermal and subcutaneousadministration.

For other routes of administration, such as by use of a perfusate,injection into an organ, or other local administration, a pharmaceuticalcomposition will be provided which results in similar levels of a tissueprotective peptide as described above. A level of about 15pM-30 nM ispreferred.

The pharmaceutical compositions of the invention may comprise atherapeutically effective amount of a compound, and a pharmaceuticallyacceptable carrier. In a specific embodiment, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized foreign pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas saline solutions in water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. A saline solution is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. The compounds ofthe invention can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin,hereby incorporated by reference herein in its entirety. Suchcompositions will contain a therapeutically effective amount of thecompound, preferably in purified form, together with a suitable amountof carrier so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

Formulations for increasing transmucosal adsorption of peptides such aslong acting tissue protective peptides are also contemplated by thecurrent invention. Pharmaceutical compositions adapted for oraladministration may be provided as capsules or tablets; as powders orgranules; as solutions, syrups or suspensions (in aqueous or non-aqueousliquids); as edible foams or whips; or as emulsions. Tablets or hardgelatine capsules may comprise lactose, starch or derivatives thereof,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,stearic acid or salts thereof. Soft gelatine capsules may comprisevegetable oils, waxes, fats, semi-solid, or liquid polyols etc.Solutions and syrups may comprise water, polyols and sugars.

An active agent intended for oral administration may be coated with oradmixed with a material that delays disintegration and/or absorption ofthe active agent in the gastrointestinal tract (e.g., glycerylmonostearate or glyceryl distearate may be used). Thus, the sustainedrelease of an active agent may be achieved over many hours and, ifnecessary, the active agent can be protected from being degraded withinthe stomach. Pharmaceutical compositions for oral administration may beformulated to facilitate release of an active agent at a particulargastrointestinal location due to specific pH or enzymatic conditions.

Pharmaceutical compositions adapted for transdermal administration maybe provided as discrete patches intended to remain intimate contact withthe epidermis of the recipient for a prolonged period of time.Pharmaceutical compositions adapted for topical administration may beprovided as ointments, creams, suspensions, lotions, powders, solutions,pastes, gels, sprays, aerosols or oils. For topical administration tothe skin, mouth, eye or other external tissues a topical ointment orcream is preferably used. When formulated in an ointment, the activeingredient may be employed with either a paraffinic or a water-miscibleointment base. Alternatively, the active ingredient may be formulated ina cream with an oil-in-water base or a water-in-oil base. Pharmaceuticalcompositions adapted for topical administration to the eye include eyedrops. In these compositions, the active ingredient can be dissolved orsuspended in a suitable carrier, e.g., in an aqueous solvent.Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouthwashes.

Pharmaceutical compositions adapted for nasal and pulmonaryadministration may comprise solid carriers such as powders (preferablyhaving a particle size in the range of 20 to 500 microns). Powders canbe administered in the manner in which snuff is taken, i.e., by rapidinhalation through the nose from a container of powder held close to thenose. Alternatively, compositions adopted for nasal administration maycomprise liquid carriers, e.g., nasal sprays or nasal drops.Alternatively, inhalation of compounds directly into the lungs may beaccomplished by inhalation deeply or installation through a mouthpieceinto the oropharynx. These compositions may comprise aqueous or oilsolutions of the active ingredient. Compositions for administration byinhalation may be supplied in specially adapted devices including, butnot limited to, pressurized aerosols, nebulizers or insufflators, whichcan be constructed so as to provide predetermined dosages of the activeingredient. In a preferred embodiment, pharmaceutical compositions ofthe invention are administered into the nasal cavity directly or intothe lungs via the nasal cavity or oropharynx.

Pharmaceutical compositions adapted for rectal administration may beprovided as suppositories or enemas. Pharmaceutical compositions adaptedfor vaginal administration may be provided as pessaries, tampons,creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injectable solutions orsuspensions, which may contain antioxidants, buffers, bacteriostats andsolutes that render the compositions substantially isotonic with theblood of an intended recipient. Other components that may be present insuch compositions include water, alcohols, polyols, glycerine andvegetable oils, for example. Compositions adapted for parenteraladministration may be presented in unit-dose or multi-dose containers,for example sealed ampules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of asterile liquid carrier, e.g., sterile saline solution for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tablets.In one embodiment, an autoinjector comprising an injectable solution ofa tissue protective peptide may be provided for emergency use byambulances, emergency rooms, and battlefield situations, and even forself-administration in a domestic setting, particularly where thepossibility of traumatic amputation may occur, such as by imprudent useof a lawn mower. The likelihood that cells and tissues in a severed footor toe will survive after reattachment may be increased by administeringa tissue protective peptide to multiple sites in the severed part assoon as practicable, even before the arrival of medical personnel onsite, or arrival of the afflicted individual with severed toe in tow atthe emergency room.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water-free concentrate in a hermetically-sealedcontainer such as an ampule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampule of sterile saline can be providedso that the ingredients may be mixed prior to administration.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

A perfusate composition may be provided for use in transplanted organbaths, for in situ perfusion, or for administration to the vasculatureof an organ donor prior to organ harvesting. Such pharmaceuticalcompositions may comprise levels of tissue protective peptides, or aform of tissue protective peptides not suitable for acute or chronic,local or systemic administration to an individual, but will serve thefunctions intended herein in a cadaver, organ bath, organ perfusate, orin situ perfusate prior to removing or reducing the levels of the tissueprotective peptide contained therein before exposing or returning thetreated organ or tissue to regular circulation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

In another embodiment, for example, a tissue protective peptide can bedelivered in a controlled-release system. For example, the peptide maybe administered using intravenous infusion, an implantable osmotic pump,a transdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRCCrit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507;Saudek et al., 1989, N. Engl. J. Med. 321:574, each of which isincorporated by reference herein in its entirety). In anotherembodiment, the compound can be delivered in a vesicle, in particular aliposome (see Langer, Science 249:1527-1533 (1990); Treat et al., inLiposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);WO 91/04014; U.S. Pat. No. 4,704,355; Lopez-Berestein, ibid., pp.317-327; see generally ibid.). In another embodiment, polymericmaterials can be used (see Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Press: Boca Raton, Fla., 1974; ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol.Sci. Rev. Macromol. Chem. 23:61, 1953; see also Levy et al., 1985,Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et at,1989, J. Neurosurg. 71:105, (each of which is incorporated by referenceherein in its entirety).

In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the target cells, tissue ororgan, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, pp. 115-138 in Medical Applications of Controlled Release, vol.2, supra, 1984, which is incorporated by reference herein in itsentirety). Other controlled release systems are discussed in the reviewby Langer (1990, Science 249:1527-1533, which is incorporated byreference herein in its entirety).

In another embodiment, tissue protective peptide, as properlyformulated, can be administered by nasal, oral, rectal, vaginal, ocular,transdermal, parenteral or sublingual administration.

In a specific embodiment, it may be desirable to administer a tissueprotective peptide of the invention locally to the area in need oftreatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as silastic membranes, or fibers. Anon-limiting example of such an embodiment would be a coronary stentcoated with a tissue protective peptide of the present invention.

Selection of the preferred effective dose will be readily determinableby a skilled artisan based upon considering several factors, which willbe known to one of ordinary skill in the art. Such factors include theparticular form of tissue protective peptide, and its pharmacokineticparameters such as bioavailability, metabolism, half-life, etc., whichwill have been established during the usual development procedurestypically employed in obtaining regulatory approval for a pharmaceuticalcompound. Further factors in considering the dose include the conditionor disease to be treated or the benefit to be achieved in a normalindividual, the body mass of the patient, the route of administration,whether administration is acute or chronic, concomitant medications, andother factors well known to affect the efficacy of administeredpharmaceutical agents. Thus the precise dosage should be decidedaccording to the judgment of the practitioner and each patient'scircumstances, e.g., depending upon the condition and the immune statusof the individual patient, and according to standard clinicaltechniques.

In another aspect of the present invention, a pharmaceutical compositionaccording to the present invention may include a tissue protectivepeptide in a formulation with at least one small molecule that exhibitstissue protective functionality. Suitable small molecules include, butare not limited to, steroids (e.g., lazaroids and glucocorticoids),antioxidants (e.g., coenzyme Q₁₀, alpha lipoic acid, and NADH),anticatabolic enzymes (e.g., glutathione peroxidase, superoxidedimutase, catalase, synthetic catalytic scavengers, as well asmimetics), indole derivatives (e.g., indoleamines, carbazoles, andcarbolines), nitric acid neutralizing agents, adenosine/adenosineagonists, phytochemicals (flavanoids), herbal extracts (ginko biloba andturmeric), vitamins (vitamins A, E, and C), oxidase electron acceptorinhibitors (e.g., xanthine oxidase electron inhibitors), minerals (e.g.,copper, zinc, and magnesium), non-steriodal anti-inflammatory drugs(e.g., aspirin, naproxen, and ibuprofen), and combinations thereof.Additionally agents including, but not limited to, anti-inflammatoryagents (e.g., corticosteroids, prednisone and hydrocortisone),glucocorticoids, steroids, non-steriodal anti-inflammatory drugs (e.g.,aspirin, ibuprofen, diclofenac, and COX-2 inhibitors), beta-agonists,anticholinergic agents and methyl xanthines), immunomodulatory agents(e.g., small organic molecules, T cell receptor modulators, cytokinereceptor modulators, T-cell depleting agents, cytokine antagonists,monokine antagonists, lymphocyte inhibitors, or anti-cancer agents),gold injections, sulphasalazine, penicillamine, anti-angiogenic agents(e.g., angiostatin), TNF-α antagonists (e.g., anti-TNFα antibodies), andendostatin), dapsone, psoralens (e.g., methoxalen and trioxsalen),anti-malarial agents (e.g., hydroxychloroquine), anti-viral agents, andantibiotics (e.g., erythromycin and penicillin) may be used inconjunction with the current pharmaceutical compositions.

In another aspect of the invention, a perfusate or perfusion solution isprovided for perfusion and storage of organs for transplant, theperfusion solution includes an amount of a tissue protective peptideeffective to protect responsive cells and associated cells, tissues ororgans. Transplant includes but is not limited to allotransplantation,where an organ (including cells, tissue or other bodily part) isharvested from one donor and transplanted into a different recipient,both being of the same species; autotransplantation, where the organ istaken from one part of a body and replaced at another, including benchsurgical procedures, in which an organ may be removed, and while exvivo, resected, repaired, or otherwise manipulated, such as for tumorremoval, and then returned to the original location orxenotransplantation, where tissues or organs or transplanted betweenspecies. In one embodiment, the perfusion solution is the University ofWisconsin (UW) solution (U.S. Pat. No. 4,798,824, hereby incorporated byreference herein in its entirety) which contains from about 1 to about25 U/ml (10 ng=1U) of tissue protective peptide, 5% hydroxyethyl starch(having a molecular weight of from about 200,000 to about 300,000 andsubstantially free of ethylene glycol, ethylene chlorohydrin, sodiumchloride and acetone); 25 mM KH₂PO₄; 3 mM glutathione; 5 mM adenosine;10 mM glucose; 10 mM HEPES buffer; 5 mM magnesium gluconate; 1.5 mMCaCl₂; 105 mM sodium gluconate; 200,000 units penicillin; 40 unitsinsulin; 16 mg dexamethasone; 12 mg Phenol Red; and has a pH of 7.4-7.5and an osmolality of about 320 mOsm/l. The solution is used to maintaincadaveric kidneys and pancreases prior to transplant. Using thesolution, preservation can be extended beyond the 30-hour limitrecommended for cadaveric kidney preservation. This particular perfusateis merely illustrative of a number of such solutions that can be adaptedfor the present use by inclusion of an effective amount of a tissueprotective peptide. In a further embodiment, the perfusate solutioncontains from about 1 to about 500 ng/ml of a tissue protective peptide,or from about 40 to about 320 ng/ml tissue protective peptide. Asmentioned above, any form of tissue protective peptide can be used inthis aspect of the invention.

While the preferred recipient of a tissue protective peptide for thepurposes herein throughout is a human, the methods herein apply equallyto other mammals, particularly domesticated animals, livestock,companion, and zoo animals. However, the invention is not so limitingand the benefits can be applied to any mammal.

In further aspects of the ex-vivo invention, any tissue protectivepeptide such as but not limited to the ones described above may beemployed.

In another aspect of the invention, methods and compositions forenhancing the viability of cells, tissues or organs which are notisolated from the vasculature by an endothelial cell barrier areprovided by exposing the cells, tissue or organs directly to apharmaceutical composition comprising a tissue protective peptide, oradministering or contacting a pharmaceutical composition containing atissue protective peptide to the vasculature of the tissue or organ.Enhanced activity of responsive cells in the treated tissue or organ isresponsible for the positive effects exerted.

Similar to other tissue protective compounds based on erythropoietin, itis possible that the tissue protective peptides of the present inventionmay be transported from the luminal surface to the basement membranesurface of endothelial cells of the capillaries of organs withendothelial cell tight junctions, including, for example, the brain,retina, and testis. Thus, responsive cells across the barrier may besusceptible targets for the beneficial effects of tissue protectivepeptides, and others cell types or tissues or organs that contain anddepend in whole or in part on responsive cells therein may be targetsfor the methods of the invention. While not wishing to be bound by anyparticular theory, after transcytosis of the tissue protective peptidemay interact with an tissue-protective receptor on a responsive cell,for example, neuronal, eye (e.g., retinal), adipose, connective, hair,tooth, mucosal, pancreatic, endocrine, aural, epithelial, skin, muscle,heart, lung, liver, kidney, small intestine, adrenal (e.g. adrenalcortex, adrenal medulla), capillary, endothelial, testes, ovary, orendometrial cell, and receptor binding can initiate a signaltransduction cascade resulting in the activation of a gene expressionprogram within the responsive cell or tissue, resulting in theprotection of the cell or tissue, or organ, from damage, such as bytoxins, chemotherapeutic agents, radiation therapy, hypoxia, etc. Inanother embodiment, the tissue protective peptide can be cross-linked toa compound that can cross the barrier, such as carbamylatederythropoietin, to be transported across the barrier in accordance withthe teaching of PCT Application No. PCT/US01/49479, U.S. patentapplication Ser. Nos. 10/188,905 and 10/185,841, incorporated herein byreference. Thus, methods for protecting responsive cell-containingtissue from injury or hypoxic stress, and enhancing the function of suchtissue are described in detail herein below.

In the practice of one embodiment of the invention, a mammalian patientis undergoing systemic chemotherapy for cancer treatment, includingradiation therapy, which commonly has adverse effects such as nerve,lung, heart, ovarian or testicular damage. Administration of apharmaceutical composition comprising a tissue protective peptide asdescribed above is performed prior to and during chemotherapy and/orradiation therapy, to protect various tissues and organs from damage bythe chemotherapeutic agent, such as to protect the testes. Treatment maybe continued until circulating levels of the chemotherapeutic agent havefallen below a level of potential danger to the mammalian body.

In the practice of another embodiment of the invention, various organsare planned to be harvested from a victim of an automobile accident fortransplant into a number of recipients, some of which required transportfor an extended distance and period of time. Prior to organ harvesting,the donor is infused with a pharmaceutical composition comprising tissueprotective peptides as described herein. Harvested organs for shipmentare perfused with a perfusate containing tissue protective peptides asdescribed herein, and stored in a bath comprising tissue protectivepeptides. Certain organs are continuously perfused with a pulsatileperfusion device, utilizing a perfusate containing tissue protectivepeptides in accordance with the present invention. Minimal deteriorationof organ function occurs during the transport and upon implant andreperfusion of the organs in situ.

In another embodiment of the present invention, a participant in ahazardous activity, one could take a dose of a pharmaceuticalcomposition containing a tissue protective peptide sufficient to eitherprevent (i.e. delaying the onset of, inhibiting, or stopping), protectagainst, or mitigate the damage resulting from an injury to a responsivecell, tissue, or organ. In particular, this method of treatment may haveapplication in various professions susceptible to injury such as, butnot limited to, professional athletes (divers, race car drivers,football players, etc.), military personnel (soldiers, paratroopers),emergency personnel (police, fire, EMS, and disaster relief personnel),stuntmen, and construction workers. Additionally, the prophylactic useof tissue protective peptides is contemplated in such recreationalendeavors including, but not limited to, rock climbing, rappelling, skydiving, racing, bicycling, football, rugby, baseball, and diving thatpose a risk of injury.

In another embodiment of the invention, a surgical procedure to repair aheart valve requires temporary cardioplegia and arterial occlusion.Prior to surgery, the patient is infused with a tissue protectivepeptide. Such treatment prevents hypoxic ischemic cellular damage,particularly after reperfusion. Additionally, the pharmaceuticalcompositions of the present invention may be used prophylactically toprepare an individual for surgery in an effort to limit the traumaassociated with the surgical procedure or aide in the recovery of theindividual from the surgical procedure. Although the present method oftreatment using pharmaceutical compositions containing tissue protectivepeptides provides a prophylactic use for surgical procedures, it may beparticularly useful in procedures that induce temporary ischemic eventsincluding, but not limited to, bypass procedures (coronary bypass),angioplasty procedures, amputations, and transplantations, as well as,those performed directly upon responsive cells, tissues, or organs suchas brain and spinal cord surgery, and open heart procedures. Suchprocedures may involve the use of cardiopulmonary (heart lung) bypass.

In another embodiment of the invention, in any surgical procedure, suchas in cardiopulmonary bypass surgery, a tissue protective peptide of theinvention can be used. In one embodiment, administration of apharmaceutical composition comprising tissue protective peptides asdescribed above is performed prior to, during, and/or following thebypass procedure, to protect the function of brain, heart, and otherorgans.

In the foregoing examples in which a tissue protective peptide of theinvention is used for ex-vivo applications, or for in vivo applicationsto treat responsive cells such as neuronal tissue, retinal tissue,heart, lung, liver, kidney, small intestine, adrenal cortex, adrenalmedulla, capillary endothelial, testes, ovary, or endometrial cells ortissue, the invention provides a pharmaceutical composition in dosageunit form adapted for protection or enhancement of responsive cells,tissues or organs distal to the vasculature which comprises an amountwithin the range from about 0.01 pg to 7.5 mg, 0.5 pg to 6.5 mg, 1 pg to5 mg, 500 pg to 5 mg, 1 ng to 5 mg, 500 ng to 5 mg, 1 μg to 5 mg, 500 μgto 5 mg, or 1 mg to 5 mg of a tissue protective peptide, and apharmaceutically acceptable carrier. In a preferred embodiment, theamount of tissue protective peptide is within the range from about 0.5pg to 1 mg. In a preferred embodiment, the formulation contains tissueprotective peptides that are non-erythropoietic.

In a further aspect of the invention, administration of tissueprotective peptides may be used to restore cognitive function in mammalshaving undergone brain trauma. After a delay of either 5 days or 30days, administration of tissue protective peptides should be able torestore function as compared to placebo-treated mammals, indicating theability of the tissue protective peptide to regenerate or restore brainactivity. Thus, the invention is also directed to the use of tissueprotective peptides for the preparation of a pharmaceutical compositionfor the treatment of brain trauma and other cognitive dysfunctions,including treatment well after the injury (e.g. three days, five days, aweek, a month, or longer). The invention is also directed to a methodfor the treatment of cognitive dysfunction following injury byadministering an effective amount of tissue protective peptides. Anytissue protective peptide as described herein may be used for thisaspect of the invention.

Furthermore, this restorative aspect of the invention is directed to theuse of any tissue protective peptides herein for the preparation of apharmaceutical composition for the restoration of cellular, tissue ororgan dysfunction, wherein treatment is initiated after, and well after,the initial insult responsible for the dysfunction. Moreover, treatmentusing tissue protective peptides of the invention can span the course ofthe disease or condition during the acute phase as well as a chronicphase.

A tissue protective peptide of the invention may be administeredsystemically at a dosage between about 1 ng and about 100 μg/kg bodyweight, preferably about 5-50 μg/kg-body weight, most preferably about10-30 μg/kg-body weight, per administration. This effective dose shouldbe sufficient to achieve serum levels of tissue protective peptidesgreater than about 80, 120, or 160 ng/ml of serum after administration.Such serum levels may be achieved at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 hours post-administration. Such doses may be repeated as necessary.For example, administration may be repeated daily, as long as clinicallynecessary, or after an appropriate interval, e.g., every 1 to 12 weeks,preferably, every 1 to 3 weeks. In one embodiment, the effective amountof tissue protective peptide and a pharmaceutically acceptable carriernosy be packaged in a single dose vial or other container. In anotherembodiment, the tissue protective peptides, which are capable ofexerting the activities described herein but not causing an increase inhemoglobin concentration or hematocrit, are used. Such tissue protectivepeptides are preferred in instances wherein the methods of the presentinvention are intended to be provided chronically.

5.4 Transcytosis

Carrier Molecule and Tissue Protective Peptide.

The present invention is further directed to a method for facilitatingthe transport of a Tissue Protective Peptide across an endothelial cellbarrier in a mammal by administering a composition which comprises thetissue protective peptide in association with a carrier peptide, apeptide capable of crossing an endothelial cell barrier, such aserythropoietin, as described hereinabove. Tight junctions betweenendothelial cells in certain organs in the body create a barrier to theentry of certain molecules. For treatment of various conditions withinthe barriered organ, means for facilitating passage of the tissueprotective peptide may be desired.

Tissue protective Peptide as Carrier Molecule.

Tissue protective peptides of the invention may be useful as carriersfor delivering other molecules across the blood-brain and other similarbarriers which they can travel across. A composition comprising amolecule desirous of crossing the barrier with a tissue protectivepeptide is prepared and peripheral administration of the compositionresults in the transcytosis of the composition across the barrier. Theassociation between the molecule to be transported across the barrierand the tissue protective peptide may be a labile covalent bond, inwhich case the molecule is released from association with the tissueprotective peptide after crossing the barrier. If the desiredpharmacological activity of the molecule is maintained or unaffected byassociation with tissue protective peptides, such a complex can beadministered.

The skilled artisan will be aware of various means for associatingmolecules with tissue protective peptides of the invention and the otheragents described above, by covalent, non-covalent, and other means.Furthermore, evaluation of the efficacy of the composition can bereadily determined in an experimental system. Association of moleculeswith tissue protective peptides may be achieved by any number of means,including labile, covalent binding, cross-linking, etc. Biotin/avidininteractions may be employed; for example, a biotinylated tissueprotective peptides of the invention may be complexed with a labileconjugate of avidin and a molecule desirably transported. As mentionedabove, a hybrid molecule may be prepared by recombinant or syntheticmeans, for example, a fusion or chimeric polypeptide which includes boththe domain of the molecule with desired pharmacological activity and thedomain responsible for the peptides tissue-protective receptor activitymodulation. Protease cleavage sites may be included in the molecule.

A molecule may be conjugated to a tissue protective peptide of theinvention through a polyfunctional molecule, i.e., a polyfunctionalcrosslinker. As used herein, the term “polyfunctional molecule”encompasses molecules having one functional group that can react morethan one time in succession, such as formaldehyde, as well as moleculeswith more than one reactive group. As used herein, the term “reactivegroup” refers to a functional group on the crosslinker that reacts witha functional group on a molecule (e.g., peptide, protein, carbohydrate,nucleic acid, particularly a hormone, antibiotic, or anti-cancer agentto be delivered across an endothelial cell barrier) so as to form acovalent bond between the cross-linker and that molecule. The term“functional group” retains its standard meaning in organic chemistry.The polyfunctional molecules that can be used are preferablybiocompatible linkers, i.e., they are noncarcinogenic, nontoxic, andsubstantially non-immunogenic in vivo. Polyfunctional cross-linkers suchas those known in the art and described herein can be readily tested inanimal models to determine their biocompatibility. The polyfunctionalmolecule is preferably bifunctional. As used herein, the term“bifunctional molecule” refers to a molecule with two reactive groups.The bifunctional molecule may be heterobifunctional or homobifunctional.A heterobifunctional cross-linker allows for vectorial conjugation. Itis particularly preferred for the polyfunctional molecule to besufficiently soluble in water for the cross-linking reactions to occurin aqueous solutions such as in aqueous solutions buffered at pH 6 to 8,and for the resulting conjugate to remain water soluble for moreeffective bio-distribution. Typically, the polyfunctional moleculecovalently bonds with an amino or a sulfhydryl functional group.However, polyfunctional molecules reactive with other functional groups,such as carboxylic acids or hydroxyl groups, are contemplated in thepresent invention.

The homobifunctional molecules have at least two reactive functionalgroups, which are the same. The reactive functional groups on ahomobifunctional molecule include, for example, aldehyde groups andactive ester groups. Homobifunctional molecules having aldehyde groupsinclude, for example, glutaraldehyde and subaraldehyde. The use ofglutaraldehyde as a cross-linking agent was disclosed by Poznansky etal., Science 223, 1304-1306 (1984). Homobifunctional molecules having atleast two active ester units include esters of dicarboxylic acids andN-hydroxysuccinimide. Some examples of such N-succinimidyl estersinclude disuccinimidyl suberate and dithio-bis-(succinimidylpropionate), and their soluble bis-sulfonic acid and bis-sulfonate saltssuch as their sodium and potassium salts. These homobifunctionalreagents are available from Pierce, Rockford, Ill.

The heterobifunctional molecules have at least two different reactivegroups. The reactive groups react with different functional groups,e.g., present on the peptide and the molecule. These two differentfunctional groups that react with the reactive group on theheterobifunctional cross-linker are usually art amino group, e.g., theepsilon amino group of lysine; a sulfhydryl group, e.g., the thiol groupof cysteine; a carboxylic acid, e.g., the carboxylate on aspartic acid;or a hydroxyl group, e.g., the hydroxyl group on serine.

Of course, certain of the various tissue protective peptides of theinvention, may not have suitable reactive groups available for use withcertain cross-linking agent; however, one of skill in the art will beamply aware of the choice of cross-linking agents based on the availablegroups for cross-linking in tissue protective peptides of the invention.

When a reactive group of a heterobifunctional molecule forms a covalentbond with an amino group, the covalent bond will usually be an amido orimido bond. The reactive group that forms a covalent bond with an aminogroup may, for example, be an activated carboxylate group, ahalocarbonyl group, or an ester group. The preferred halocarbonyl groupis a chlorocarbonyl group. The ester groups are preferably reactiveester groups such as, for example, an N-hydroxy-succinimide ester group.

The other functional group typically is either a thiol group, a groupcapable of being converted into a thiol group, or a group that forms acovalent bond with a thiol group. The covalent bond will usually be athioether bond or a disulfide. The reactive group that forms a covalentbond with a thiol group may, for example, be a double bond that reactswith thiol groups or an activated disulfide. A reactive group containinga double bond capable of reacting with a thiol group is the maleimidogroup, although others, such as acrylonitrile, are also possible. Areactive disulfide group may, for example, be a 2-pyridyldithio group ora 5, 5′-dithio-bis-(2-nitrobenzoic acid) group. Some examples ofheterobifunctional reagents containing reactive disulfide bonds includeN-succinimidyl 3-(2-pyridyl-dithio)propionate (Carlsson, et al., 1978,Biochem J., 173:723-737), sodiumS-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene.N-succinimidyl 3-(2-pyridyldithio)propionate is preferred. Some examplesof heterobifunctional reagents comprising reactive groups having adouble bond that reacts with a thiol group include succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate and succinimidylm-maleimidobenzoate.

Other heterobifunctional molecules include succinimidyl 3-(maleimido)propionate, sulfosuccinimidyl 4-(p-maleimido-phenyl)butyrate,sulfosuccinimidyl 4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,maleimidobenzoyl-N-hydroxy-succinimide ester. The sodium sulfonate saltof succinimidyl m-maleimidobenzoate is preferred. Many of theabove-mentioned heterobifunctional reagents and their sulfonate saltsare available from Pierce Chemical Co., Rockford, Ill. USA.

The need for the above-described conjugated to be reversible or labilemay be readily determined by the skilled artisan. A conjugate may betested in vitro for desirable pharmacological activity. If the conjugateretains both properties (the properties of the conjugated molecule andthe properties of the tissue protective peptide), its suitability maythen be tested in vivo. If the conjugated molecule requires separationfrom the tissue protective peptide for activity, a labile bond orreversible association with long acting erythropoietin or the longacting tissue protective cytokine will be preferable. The labilitycharacteristics may also be tested using standard in vitro proceduresbefore in vivo testing.

Additional information regarding how to make and use these as well asother polyfunctional reagents may be obtained from the followingpublications or others available in the art:

-   Carlsson, J. et al., 1978, Biochem. J. 173:723-737;-   Cumber, J. A. et al., 1985, Methods in Enzymology 112:207-224;-   Joe, R. et al., 1978, Biochem 17:5399-5405;-   Sun, T. T. et al., 1974, Biochem. 13:2334-2340;-   Blattler, W. A. et al., 1985, Biochem. 24:1517-152;-   Liu, F. T. et al., 1979, Biochem. 18:690-697;-   Youle, R. J. and Neville, D. M. Jr., 1980, Proc. Natl. Acad. Sci.    U.S.A. 77:5483-5486;-   Lerner, R. A. et al., 1981, Proc. Natl. Acad. Sci. U.S.A.    78:3403-3407;-   Jung, S. M. and Moroi, M., 1983, Biochem. Biophys. Acta 761:162;-   Caulfield, M. P. et al., 1984, Biochem. 81:7772-7776;-   Staros, J. V., 1982, Biochem. 21:3950-3955;-   Yoshitake, S. et al., 1979, Eur. J. Biochem. 101:395-399;-   Yoshitake, S. et al., 1982, J. Biochem. 92:1413-1424;-   Pilch, P. F. and Czech, M. P., 1979, J. Biol. Chem. 254:3375-3381;-   Novick, D. et al., 1987, J. Biol. Chem. 262:8483-8487;-   Lomant, A. J. and Fairbanks, G., 1976, J. Mol. Biol. 104:243-261;-   Hamada, H. and Tsuruo, T., 1987, Anal. Biochem. 160:483-488; or-   Hashida, S. et al., 1984, J. Applied Biochem. 6:56-63, each of which    is hereby incorporated by reference in its entirety.

Additionally, methods of cross-linking are reviewed by Means and Feeney,1990, Bioconjugate Chem. 1:2-12, hereby incorporated by reference in itsentirety.

Barriers which are crossed by the above-described methods andcompositions of the present invention include but are not limited to theblood-brain bather, the blood-eye barrier, the blood-testes barrier, theblood-ovary barrier, blood-nerve barrier, blood-spinal cord barrier, andblood-placenta barrier.

Candidate molecules for transport across an endothelial cell barrierinclude, for example, hormones, such as growth hormone, neurotrophicfactors, antibiotics, antivirals, or antifungals such as those normallyexcluded from the brain and other barriered organs, peptideradiopharmaceuticals, antisense drugs, antibodies and antivirals againstbiologically-active agents, pharmaceuticals, and anti-cancer agents.Non-limiting examples of such molecules include hormones such as growthhormone, nerve growth factor (NGF), brain-derived neurotrophic factor(BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growthfactor (bFGF), transforming growth factor β1 (TGFβ1), transforminggrowth factor β2 (TGFβ2), transforming growth factor β3 (TGFβ3),interleukin 1, interleukin 2, interleukin 3, and interleukin 6, AZT,antibodies against tumor necrosis factor, and immunosuppressive agentssuch as cyclosporin. Additionally, dyes or markers may be attached tothe tissue protective peptides of the present invention in order tovisualize cells, tissues, or organs within the brain and other barrieredorgans for diagnostic purposes. As an example, a marker used tovisualize plaque within the brain could be attached to a tissueprotective peptide in order to determine the progression of Alzheimer'sdisease within a patient.

The present invention is also directed to a composition comprising amolecule to be transported via transcytosis across an endothelial celltight junction barrier and a tissue protective peptide as describedabove. The invention is further directed to the use of a conjugatebetween a molecule and a tissue protective peptide cytokine as describedabove for the preparation of a pharmaceutical composition for thedelivery of the molecule across a barrier as described above.

Various animal models and in-vitro tests of neuroprotection andtranscytosis are provided in PCT/US01/49479 (hereby incorporated byreference herein in its entirety) to demonstrate the effectiveness ofthe tissue protective peptides of the invention. For transcytosis, modelproteins conjugated to the long acting erythropoietins of the inventionare evaluated for transport into the brain following parenteraladministration. These tests in in-vitro models and animal models arepredictive of the efficacy of the present compounds in other mammalianspecies including humans.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

6. EXAMPLES Example 1 Method of Peptide Synthesis

A. Synthesis of Peptide A (SEQ ID NO:32, corresponding to EPO amino acidsequence 38-57) and Peptide B (SEQ ID NO:34, corresponding to EPO aminoacid sequence 58-82).

Peptide A, SEQ ID NO:32, and Peptide B, SEQ ID NO:34, fragments of EPO(see Table 1), were synthesized using “in situ neutralization” BocChemistry stepwise solid-phase peptide synthesis, as described in Band,D., Chopra, N. and Kent, S., “Total Synthesis of Crambin,” J. AM. CHEM.SOC. 2004, 126, 1377-1383 (incorporated by reference herein in itsentirety). Briefly, two fragments corresponding to EPO amino acidsequence 38-57 (peptide C, NITVPDTKVNFYAWKRMEVG, SEQ ID NO:29) and EPOamino acid sequence 58-82 (peptide D, QQAVEVWQGLALLSEAVLRGQALLV, SEQ IDNO:30) were synthesized on —OCH₂-Pam-resins (free ^(α)carboxyl peptides)or on HSCH₂CH₂CO-Leu-OCH₂-Pam-Resin (^(α)Thioester peptides). Duringsynthesis the side chains of various amino acids were protected asfollows: Arg(Tos), Asn(Xan), Asp(OcHex), Cys(4-CH₃Bzl) or Cys(ACM),Glu(OcHex), Lys(2-Cl—Z), Ser(Bzl), Thr(Bzl), Tyr(Br—Z). After thepeptide chain was assembled, the peptides were deprotected andsimultaneously cleaved from the resin support by treatment withanhydrous HF containing p-cresol (90:10, v/v) for 1 hr at 0° C. Afterevaporation of the HF under reduced pressure, crude products wereprecipitated and triturated with chilled diethyl ether, and the peptideswere dissolved in 50% aqueous acetonitrile containing 0.1% TFA andpurified by the preparative HPLC system. Peptide compositions wereconfirmed using LC-MS.

Example 2 Validation of Peptide-Mediated Tissue Protection

The tissue protective peptides were tested for any tissue protectiveactivity using a Sciatic Nerve Assay. Sprague-Dawley rats (250-300grams) (six per group, including control) were anesthetized usingisoflurane (Baxter NPC 10019-773-60) and a Table Top LaboratoryAnesthesia System (flowmeter set to 2-3 liters/minute @ 55 psi) for atleast 3 minutes. The rat was then placed on a homeothermic blanket toensure that the core temperature of the rat was maintained at 35-37° C.during the operation. Core temperature was monitored via a rectal probe.The right sciatic nerve of the anesthetized rat was exposed at mid thighthrough a quadriceps muscle dissection; a 2 cm incision with a 15 bladescalpel was made through the skin parallel and over the quadricepsmuscle and the quadriceps muscle was cut to expose the sciatic nerveusing a pair of dissecting scissors. The sciatic nerve was then freedfrom the surrounding membranes. A 2-0 braided silk thread (Ethicon,685-G) was passed under the nerve and the ends of the suture passedthrough a guide which was maintained perpendicular to the nerve. The endof the suture was then tied to a non-elastic cord which was then drapedaround the pulley system (a NYL pulley bearing MTD ¼″B (PO Number04174-01) with stabilizer) and a 100 gram weight attached to thenon-elastic cord was slowly released. The weight was allowed to hang for1 minute before the silk suture was cut to release the weight.

A 289 pmol/kg dose of carbamylated erythropoietin, a 289 pmol/kg dose ofone peptide from the series A-J (see Table 1), or PBS was then injectedinto the caudal vein using ½ cc insulin syringe. A 20 mer fragment(corresponding to amino acids 102-121) from pigment epithelium-derivedgrowth factor (PEDF) which does not follow the teaching above was usedas control.

The muscle and surgical incision were then closed and 5 ml of LactatedRingers solution was injected subcutaneously into the rat. The coretemperature of the rat was maintained at 35-37° C. using a heat blanketduring recovery.

Over the next four days the rear toe splaying of the rats was determinedby placing the rat in an acrylic tube with a diameter of 30 cm on thescanning surface of a digital scanner. After waiting 5 minutes in orderto permit acclimation, a scan was taken of the rat's back feet thatclearly displayed all 5 toes. Three acceptable scans of each rat weretaken. From the scans, the Toe Spread (the distance between the ball ofthe first toe and the ball of the fifth toe) and the Intermediate ToeSpread (the distance between the ball of the second toe and the ball ofthe fourth toe) were measured. The static sciatic index was thencomputed in accordance with S. Erbayraktar et at, 2003, Proc Natl AcadSci USA 100, 6741-6746 (hereby incorporated by reference in itsentirety) and statistical analysis performed.

All peptides except B (SEQ ID NO:34), H (SEQ ID NO:47) and the PEDFderivative were equally protective, providing a static sciatic index(“SSI”) of about −0.57 versus a SSI of about −67 to about −68 forPBS/PEDF fragment (FIG. 2). FIG. 2 also shows that the efficacy of thepositive peptides was at least equivalent, if not improved, over that ofthe carbamylated erythropoietin.

Table I also presents the approximate distance between carbonyl carbonsfor the tested peptides. Distances were calculated using thethree-dimensional coordinates provided by Cheetham et al., 1998, Nat.Struct. Biol. 5:861-866, hereby incorporated by reference. The peptideswhich tested positive for tissue protective activity each had a carbonylcarbon to carbonyl carbon distance/separation of between about 3 Å toabout 5 Å.

TABLE 1 Tissue protective efficacy of representative peptides using anin vivo bioassay (sciatic nerve injury model). Approx DistanceBetween carbonyl Dose Sciatic EPO Carbons [nmoles/ nerve Peptide Classpeptide sequence Structure (Angstroms) kg-bw] assay A) EPO A  1-23APPRLICDSRVLERYLLEAKEAE 4.6  29, 290, 1450 + fragment (SEQ ID NO: 32)APPRLICDSRVLERYLLEAKEAE 4.4 (SEQ ID NO: 32) B 24-37 NITTGCAEHCSLNE 2.8290 − (SEQ ID NO: 34) C 38-57 NITVPDTKVNFYAWKRMEVG 4.6 290 +(SEQ ID NO: 29) D 58-82 QQAVEVWQGLALLSEAVLRGQALLV 4.8  29, 290, 1450 +(SEQ ID NO: 30) E 28-47 GCAEHCSLNENITVPDTKVN 4.4 290 + (SEQ ID NO: 31) F14-29 RYLLEAKEAENITTGC 3.6 290 + (SEQ ID NO: 33) B) Helix face G58, 62, 65, 69, QEQLERALNSS 3.6 290 + 72, 76, 79, 80, (SEQ ID NO: 40)83, 84, 85 H 71, 72, 75, 76, SELRGQ 7.2 290 − 77 (SEQ ID NO: 47)C) chimera I Peptide G + CSLNENIQEQLERALNSS 290 + β-pleated(SEQ ID NO: 43) sheet (33-39) J Peptide G + QEQLERALNSSLRRYINMLTRTR290 + pancreatic (SEQ ID NO: 41) polypeptide helix D) Type 1 K GM-CSFWEHVNAIQEARRLL 3.6 290 + cytokine motif helix A (13-26) (SEQ ID NO: 35)WEHVNAIQEARRLL 4.6 (SEQ ID NO: 35) WEHVNAIQEARRLL 4.6 (SEQ ID NO: 35) LCNTF helix KIRSDLTALTESYVKH 4.7 290 + A (26-41) (SEQ ID NO: 37)

Example 3 Tissue Protective Peptides are Non Erythropoietic A. In VitroAssessment:

UT-7epo, a human erythropoietin-dependent leukemia cell line, was usedfor the determination of the erythropoietic potency of the peptides.UT-7epo cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ), Cat. No. ACC 363) were grown in a complete RPMI-1640 medium with10% FBS and 5 ng/ml erythropoietin. The proliferation/survival(=viability increase) response of the cells exposed to erythropoietin ismediated by the classical erythrocyte-type erythropoietin receptor andis a quantitative measure of the capacity of erythropoietin-variants tostimulate the classical erythropoietin receptor.

UT-7epo cells were transferred to fresh complete RPMI 1640 mediumcontaining 10% donor calf serum, 4 mM L-glutamine, and supplemented with5 ng/ml of recombinant human erythropoietin. The cells were maintainedin 75 cm² flasks with 20 ml of medium/flask in a humidified incubatorwith 5% CO₂ at 37° C. for 48 h. On day two of the assay, i.e., at 48 h,the cells were transferred from the flask into a 50-ml conical tube andcentrifuged at 1,000 rpm for 5 minutes at room temperature. Thesupernatant was discarded and the cells washed two times with 10 ml ofstarvation media (3% donor calf serum, 4 mM L-glutamine). The cells werethen re-suspended in starvation media, using up and down pipette actionto obtain a single cell suspension. The re-suspended cells were dilutedwith starvation media to a obtain a density of 4×10⁵ cells/ml, andplated at a total culture volume of 10 ml per 25 cm² flask. Following a4 h incubation, the cells were again transferred to a 50-ml conicaltube. Control cells were maintained throughout with 5 ng/ml ofrhu-erythropoietin.

Cells were diluted to 200,000 cells/ml in starvation medium, plated at100 μl/well in a 96 well plate and exposed to varying concentrations oferythropoietin, carbamylated erythropoietin, and Peptide D, SEQ IDNO:30. A series of 10 fold dilutions in RPMI1640 medium containing 3%serum was used to generate concentrations of test compounds from 0.2 pMto 20 nM. Following a further for 48 h incubation, a solution of 15 mlWST-1 Cell Proliferation Reagent (Roche) was added to each well, andincubated for 1 hour at 37° C. in CO₂. After mixing for 1 minute, theplate was read in a plate reader (absorption at 450 nm, subtracted frombackground absorption at 650 nm).

Peptide D exhibited no erythropoietic activity at doses as high as10,000 μM (FIG. 3). Preferably, the peptide will have no erythropoieticactivity for a dose lower than 1 μg/ml, and more preferably for a doselower than 10 μg/ml.

B. In Vivo Assessment:

To evaluate the erythropoietic activity of tissue protective peptide F(SEQ ID NO:33) or peptide G (SEQ ID NO:40, as discussed supra a peptideconstructed of the presenting residues of Helix B), the peptides wereadministered 0.8 μg/kg subcutaneously three time per week to maleSprague Dawley rats. The dosage schedule corresponded to the equivalentdose (on a molar basis) of EPO previously determined to be elicitmaximum erythropoiesis. Hemoglobin concentration was determinedperiodically by use of an automated analyzer (Keska Corporation).

Neither Peptide B nor Peptide C showed any increase in hematocrit overthe course of the study (FIG. 4; the response to an equimolar dosage ofEPO is presented for comparison). The decrease in hemoglobin noted forEPO after 3 weeks is due to the production of anti-EPO neutralizingantibodies which cause pure red cell aplasia. In contrast, noneutralizing antibody response was observed for either peptide G orpeptide F.

Example 4 Peptide is Tissue Protective in In Vitro Assays

Peptides can be readily assessed for tissue protection using any numberof in vitro assays. For example, protection from excitoxicity can bedetermined using kainite-induced death of mouse motoneurons. Spinalcords were obtained from 15-day old Sprague-Dawley rat embryos aspreviously described (Siren et al., 2001, PNAS 98:4044, herebyincorporated by reference in its entirety). The ventral horn wastrypsinized and centrifuged through a 4% BSA cushion for 10 min at300×g. Cells (representing mixed neuron-glia culture) were seeded at adensity of 2,000 cells/cm² into 4-mm well plates precoated with poly-DLornithine and laminin. Motoneurons were further purified byimmunopanning and the cells were seeded at low density (20,000cells/cm²) onto 24-mm well plates precoated with poly-DL-ornithine andlaminin, and containing complete culture medium [Neurobasal/B27 (2%);0.5 mM L-glutamine; 2% horse serum; 25 mM 2 mercaptoethanol; 25 mMglutamate; 1% penicillin and streptomycin; 1 ng/ml BDNF]. The medium(without glutamate) was re-added to cultures on days 4 and 6.

Cell death was induced on day 6 in culture by incubation for 48 h withkainic acid (5 mM for mixed neuron-glia cultures; 50 mM for purifiedcultures). Peptide D (5 ng/mL) or vehicle was added to the cultures 72 hbefore induction of cell death, and treatment continued for 48 h. Themedium was then discarded and the cells fixed with 4% (vol/vol)paraformaldehyde in PBS for 40 min, permeabilized with 0.2% TritonX-100, blocked with 10% (vol/vol) FCS in PBS, incubated with antibodiesagainst non-phosphorylated neurofilaments (SMI-32; 1:9,000) overnight,and visualized by using the avidin-biotin method with diaminobenzidine.Viability of motoneurons was assessed morphologically by counting SMI-32positive cells across four sides of the cover slip and staining forapoptotic bodies was done by using H33258.

Peptide D (SEQ ID NO:30, corresponding to amino acids 58-82 of SEQ IDNO:1) completely protected motoneurons from injury caused by kainate(FIG. 5).

Alternatively, tissue protection afforded by peptides can be determinedusing an assay employing mouse P19 cells, which are neuronal-like anddie via apoptosis upon withdrawal of serum. Tissue protection of peptideD (SEQ ID NO:30) was compared to that of EPO using P19 clone P19S1801A1as previously published (Siren et al., 2001, PNAS 98:4044, nerebyincorporated by reference in its entirety). Cells were maintainedundifferentiated in DMEM supplemented with 2 mM Lglutamine; 100 units/mlpenicillin G; 100 mg/ml streptomycin sulfate (GIBCO); 10% (vol/vol) FBS(HyClone), containing 1.2 g/liter NaHCO3 and 10 mM Hepes buffer,hereafter referred to as complete medium. Serum-free medium containedthe same components as above with the deletion of serum and the additionof 5 mg/ml of insulin; 100 mg/ml of transferrin; 20 nM progesterone; 100mM putrescine; 30 nM Na2SeO3 (from Sigma). For the experiments, 50%confluent cells were pretreated overnight with EPO or vehicle,dissociated with trypsin, washed in serum-free medium, and plated in25-cm² tissue culture flasks at a final density of 104 cells/cm² inserum-free medium alone or with added EPO. Cell viability and wasdetermined by trypan blue exclusion and a hemacytometer.

Peptide C, SEQ ID NO:29 (corresponding to amino acids 38-57 of SEQ IDNO:1) was at least 10 time more potent on a weight basis than EPO inpreventing apoptosis of p19 cells (FIG. 6).

Example 5 Middle Cerebral Artery Occlusion Model

Male Crl:CD(SD)BR rats weighing 250-280 g were obtained from CharlesRiver, Calco, Italy. Surgery was performed in accordance with theteachings of Brines et al., 2000, PNAS USA 97:10526-10531 (herebyincorporated by reference in its entirety. Briefly, the rats wereanesthetized with chloral hydrate (400 mg/kg-bw, i.p.), the carotidarteries were visualized, and the right carotid was occluded by twosutures and severed. A burr hole adjacent and rostral to the right orbitallowed visualization of the middle cerebral artery (“MCA”), which wascauterized distal to the rhinal artery. To produce a penumbra (borderzone) surrounding this fixed MCA lesion, the contralateral carotidartery was occluded for 1 hour by using traction provided by a fineforceps and then re-opened.

Sprague Dawley rats (8 per group) were subjected to the above noted MCAOprotocol. The rats were administered PBS, carbamylated erythropoietin(44 ug/kg), or peptide D (aa 58-82; 4.4 ug/kg) upon release of theocclusion. Additionally, peptide D (aa 58-82; 4.4 ug/kg) wasadministered in four doses at 2 hour intervals following the occlusionto a separate group. For assessment of injury, rats were subjected tobehavioral testing or the volume of the lesion was determined bytetrazolium staining of brain sections performed 24 hours post surgeryin accordance with the previously noted protocol.

FIG. 7A presents a graph demonstrating the volume of lesions resultingfrom the MCAO protocol. Treatment with peptide D (SEQ ID NO:30), eitheras a single dose or by multiple doses, reduced the lesion volumeresulting from the MCAO surgery by about two thirds: statisticallyequivalent to the tissue protective effects of carbamylatederythropoietin.

(b) Therapeutic Window of Tissue Protective Cytokines

The MCAO protocol as outlined above was repeated for the instantexample. Following the occlusion procedure, PBS, carbamylatederythropoietin (44 ug/kg, i.v.), or peptide D (SEQ ID NO:30) (4.4 ug/kg)were administered to the rats immediately after recirculation wasestablished in the carotid (i.e., one hour from the onset of ischemia).In addition, peptide D (SEQ ID NO:30) was administered in four doses(each 4.4 ug/kg-bw) at 2 hours intervals following the occlusion. (8rats per group).

(c) Behavioral Testing

A separate group of rats was also tested in a foot fault behavioralprotocol. Rats were tested on an elevated stainless steel grid floor 30cm×30 cm with grid size of 30 mm according to the protocol of Markgrafet al., 1992, Brain Research 575:238-246 (hereby incorporated byreference in its entirety). When placed on the grid, rat would attemptto move around and occasionally place a foot, rather than on the grid,through a grid opening (“foot fault”). The number of foot faults wasmeasured for a 1 minute period.

The rats treated with peptide D (SEQ ID NO:30) following reperfusionsuffered from fewer foot faults than those treated with PBS (FIG. 7B).No significant additional benefit was observed following theadministration of multiple doses of peptide D (SEQ ID NO:30). Althoughthe mean number of foot faults was less in the group receiving multipledoses of peptide, the difference observed was not significantlydifferent from the group receiving a single dose.

Example 6 Diabetic Neuropathy

Diabetes was induced in male Sprague Dawley rats (Charles River, Calco,IT) using streptozocin administered at a single dose of 60 mg/kg ip infasting rats as previously described (Bianchi et al., 2004, Proc NatlAcad Sci USA 101, 823-828, hereby incorporated by reference in itsentirety). Diabetes was confirmed by increased serum glucose levels togreater than 300 mg per deciliter (mg %) (normal levels are <100 mg %).Diabetic animals were then treated with peptide D (SEQ ID NO:30; 4μg/kg) or vehicle 5 times a week intraperitoneally. Two weeks afterinduction of the diabetic state, nerve conduction velocity wasdetermined using the caudal nerve.

As shown in FIG. 8A, the diabetic animals exhibited a reduction incaudal nerve conduction velocity from about 22 m/s (normal) to about 19m/s. Administration of peptide D (SEQ ID NO:30) was associated with anincrease in conduction velocity to about 23 m/s.

Additionally, the thermal nociceptive threshhold was quantified bymeasurement of the time to paw withdrawal in a “hot plate” test.Withdrawal latency was defined as the time between placement on the hotplate and the time of withdrawal and licking of hind paw. Each animalwas tested twice separated by a 30 min rest interval. Hind paw thermalthresh-hold was measured 4 weeks after induction of diabetes. Peptide D(SEQ ID NO:30) reduced the latency time spent on the hot plate by thediabetic animal (FIG. 8B).

Example 7 Protection of Sciatic Nerve and Kidney fromCisplatinum-Induced Injury

Cisplatinum (CDDT) was administered intraperitoneally to maleSprague-Dawley rats at 2 mg/kg twice weekly for 5 weeks as described inBianchi et al., 2006, Clin Cancer Res 12: 2607-2612, hereby incorporatedby reference in its entirety. Animals were separated into groups of 6each. During the 5 week CDDT administration, animals also receivedeither peptide G, (SEQ ID NO:40) at 0.4 μg/kg-bw or PBS i.p. three timesper week. A control group received PBS instead of CDDT. Hot platelatency was determined as described in Example 6 above.

Animals that received CDDT and only PBS exhibited an increase in latencycompared to controls: i.e., CDDT was associated with impaired thermalsensitivity. In contrast, animals that received the peptide exhibitednormal hot plate latency (FIG. 9A).

Treatment with peptide also prevented CDDT-induced polyuria (FIG. 9B)Specifically, animals that had received PBS exhibited a significantincrease in daily urine production from about 30 mL/day to about 47mL/day. In contrast, animals having received the tissue-protectivepeptide did not significantly differ from controls animals that receivedPBS instead of CDDT.

Example 8 Protection from Diabetes-Induced Retinal Vascular Leak

Beneficial effects of tissue-protective peptides onhyperglycemia-induced retinal vascular leakiness can be determined usinga rat model of diabetic retinopathy. In this model, Evans blue is usedto determine leakage from the blood vessel into tissues as described byXu et al., 2001, Invest. Ophthal. Vis. Sci 42: 789-794 (herebyincorporated by reference in its entirety). Evans blue is tightly boundto albumin and is therefore retained within the circulation unlessleakiness of the vessel wall occurs, such as caused by uncontrolleddiabetes mellitus.

In this model, fasting male Sprague-Dawley rats receive a single dose ofstreptozotocin (60 mg.kg ip). Two days later, following verification ofthe development of diabetes mellitus (fasting serum glucose greater than300 mg %, animals were divided into groups of 6 animals each as well asa control group that did not receive streptozotocin. The two diabeticgroups were administered either peptide D (SEQ ID NO:30) at 4 μg.kgintraperitoneally 5 days a week or PBS on the same schedule. After threeweeks of uncontrolled diabetes, animals were anesthetized andadministered Evans Blue dye (30 mg/kg) intravenously, which was allowedto circulate for 2 hours. Using transcardiac puncture, the animals werethen perfused with PBS until the effluent was clear followed by 4%paraformaldehyde. The eyes were then removed and the retinas carefullydissected from the globe. The retinal content of Evans Blue wasdetermined by incubating the retinas in formamide at 80° C. for 18hours. The supernatant was then removed and saved for analysis and theretinas completely dried and weighed. Concentration of Evans Blue in thesupernatant was determined by a spectrophotometer and a standard curveof Evans Blue dissolved in formamide established.

As seen in FIG. 10, animals that received administration of peptide D(SEQ ID NO:30) experienced no increase in Evans Blue dye within theretina, compared to control. In contrast, diabetic animals that receivedonly PBS exhibited an increase in retinal Evans blue content, indicatingthe vascular leakage had occurred.

Example 9 Protection from Acute Renal Failure

Tissue-protective peptides are also effective in preventing injury tothe kidneys in the setting of ischemia. Adult male Wistar rats wereanesthetized and an abdominal incision made to visualize both renalarteries. Using an atraumatic vascular clamp, both arteries werecompressed for 60 minutes, completely arresting renal blood flow. Theclamps were then removed to restore circulation and peptide F (SEQ IDNO:33) or peptide G (SEQ ID NO:40) was administered at 290 pmol/kg-bwintravenously. An additional group undergoing ischemia received only PBSintravenously.

Seventy two hours following reperfusion, the animals were anesthetizedand underwent perfusion-fixation using paraformaldehyde. Fixed animalswere sectioned sagittally into halves and further fixed by immersion in10% formaldehyde at room temperature for one day. Histologicalevaluation of the kidneys was performed according to the protocol ofSharpies et al., 2005, J Amer Soc Nephrol: 15: 2115 (hereby incorporatedby reference in its entirety). Briefly, after dehydration using gradedethanol, pieces of kidney were embedded in parafin, cut into 5micrometer sections and mounted on glass slides. Sections on slides weredeparaffinized with xylene, counterstained with hematoxylin and eosin,and examined under a light microscope. One hundred fields were examinedfor each kidney, and a score from 0 to 3 was given for each tubularprofile: 0, normal histology; 1, tubular cell swelling, brush borderloss, and nuclear condensation with up to one third nuclear loss; 2, asfor score 1 but greater than one third and less than two thirds tubularprofiles showing nuclear loss; and 3, greater than two thirds tubularprofile showing nuclear loss. The histologic score for each kidney wascalculated by addition of all scores, with a maximum score of 300.

Administration of either peptide F (SEQ ID NO:33) or peptide G (SEQ IDNO:40) was associated with a significant reduction in injury score(p<0.05) compared to the controls.

Example 11 Efficacy of Tissue Protective Peptides in Cerebral Malaria

A rodent model of cerebral malaria was developed according to Kaiser etal., 2006, J. Infect. Dis. 193:987-995 (hereby incorporated by referencein its entirety. Female CBA/J mice 7 weeks old were separated intogroups of 20 animals. Each group was infected with Plasmodium bergheiAnka (PbA) administered intraperitoneally as a dose of 10⁶ PbA infectederythrocytes. Mice received either PBS or peptide F (SEQ ID NO:33) ondays 4, 5, and 6 as intraperitoneal injection at a dose of 2.6 μg/kg.Clinical status and blood smear data were gathered during the follow-up(end point D30). Cumulative long-term survival was calculated accordingto the Kaplan-Meier method and groups were compared with the log ranktest. Survival time was the dependant variable. A p-value of <0.05 wasconsidered significant.

As shown in FIG. 12, all mice in the control group (saline) died by day8. In contrast, mice that received peptide F (SEQ ID NO:33) exhibitedprolonged survival, significantly different from the control group(p<0.005), using a log-rank test.

Example 12 Efficacy of Tissue Protective Peptides in a Murine EAE Model

Experimental autoimmune encephalomyelitis (“AEA”) was induced in C57BL/6female mice (6-8 weeks of age) according to Savino et al., 2006, JNeuroimmunol 172:27-37, hereby incorporated by reference in itsentirety. EAE was induced by subcutaneous immunization in the flankswith a total of 200 μg of MOG35-55 (Multiple Peptide Systems, San Diego,Calif., USA) in incomplete Freund's adjuvant (Sigma, St. Louis, Mo.,USA) supplemented with 8 mg/ml of Mycobacterium tuberculosis (strainH37RA; Difco, Detroit, Mich., USA). Animals were housed in specificpathogen-free conditions, allowing access to food and water ad libitum.Mice received 500 ng of pertussin toxin (Sigma) i.v. at the time ofimmunization and 48 h later. Weight and clinical score were recordeddaily (0=healthy, 1=flaccid tail, 2=ataxia, and/or hind-limbs paresis,or slow righting reflex, 28 C. 3=paralysis of hind limb and/or paresisof forelimbs, 4=paraparesis of fore limb, 5=moribund or death). Foodpellets and the drinking water were placed on Petri plates on the floorof the cage to enable sick mice to eat and drink. Peptide E (SEQ IDNO:31) was administered daily subcutaneously at a dose of 4.4micrograms/kg-bw, starting on day 4 after immunization.

Administration of peptide E (SEQ ID NO:31) significantly reduced boththe time course and severity of the clinical presentation of AEA in thetreated animals (p<0.01) (FIG. 13).

The invention is not to be limited in scope by the specific embodimentsdescribed which are intended as single illustrations of individualaspects of the invention, and functionally equivalent methods andcomponents are within the scope of the invention. Indeed variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

All references cited herein are incorporated by reference herein intheir entireties for all purposes.

1.-57. (canceled)
 58. A method for protecting or maintaining theviability of a responsive cell, tissue, or organ isolated from a mammalcomprising exposing said cell, tissue, or organ to an isolatedpolypeptide consisting of no more than 30 amino acid residues, whereinthe isolated polypeptide comprises the amino acid sequence QEQLERALNSS(SEQ ID NO: 40), and wherein one amino acid residue of SEQ ID NO: 40 isoptionally replaced with a conservative or non-conservative substitutionthat is an amino acid or amino acid equivalent.
 59. The method of claim58, wherein the isolated polypeptide consists of the amino acid sequenceQEQLERALNSS (SEQ ID NO: 40), wherein one amino acid residue of SEQ IDNO: 40 is optionally replaced with a conservative or non-conservativesubstitution that is an amino acid or amino acid equivalent.
 60. Themethod of claim 58 or 59, wherein one of the amino acid residues of SEQID NO: 40 is replaced with a conservative or non-conservativesubstitution that is an amino acid or amino acid equivalent.
 61. Themethod of claim 60, wherein the amino acid equivalent is an amino acidhaving a side chain modification or side chain substitution.
 62. Themethod of claim 58, wherein the isolated polypeptide consists of theamino acid sequence QEQLERALNSS (SEQ ID NO: 40).
 63. The method of claim58, wherein the mammal is a human.
 64. A method for the treatment of acardiovascular disease, cardiopulmonary disease, respiratory disease,kidney disease, disease of the urinary system, disease of thereproductive system, bone disease, skin disease, gastrointestinaldisease, endocrine abnormality, metabolic abnormality, cognitivedysfunction, or a disease or disorder of the central or peripheralnervous system in a subject in need thereof, said method comprisingadministering to the subject an isolated polypeptide consisting of nomore than 30 amino acid residues, wherein the isolated polypeptidecomprises the amino acid sequence QEQLERALNSS (SEQ ID NO: 40), andwherein one amino acid residue of SEQ ID NO: 40 is optionally replacedwith a conservative or non-conservative substitution that is an aminoacid or amino acid equivalent.
 65. The method of claim 64, wherein theisolated polypeptide consists of the amino acid sequence QEQLERALNSS(SEQ ID NO: 40), wherein one amino acid residue of SEQ ID NO: 40 isoptionally replaced with a conservative or non-conservative substitutionthat is an amino acid or amino acid equivalent.
 66. The method of claim64 or 65, wherein one of the amino acid residues of SEQ ID NO: 40 isreplaced with a conservative or non-conservative substitution that is anamino acid or amino acid equivalent.
 67. The method of claim 66, whereinthe amino acid equivalent is an amino acid having a side chainmodification or side chain substitution.
 68. The method of claim 64,wherein the isolated polypeptide consists of the amino acid sequenceQEQLERALNSS (SEQ ID NO: 40).
 69. The method of claim 64, wherein themethod is for the treatment of a disease or disorder of the central orperipheral nervous system.
 70. The method of claim 69, wherein themethod is for the treatment of a trauma and/or inflammation of theperipheral nerves in a subject.
 71. The method of claim 70, wherein thetrauma and/or inflammation to the peripheral nerves is associated with asciatic nerve injury, diabetic neuropathy or carpal tunnel syndrome. 72.The method of claim 64, wherein the method is for the treatment of anendocrine or metabolic abnormality in a subject.
 73. The method of claim72, wherein the endocrine or metabolic abnormality is diabetes mellitustype I or diabetes mellitus type II.
 74. The method of claim 64, whereinthe subject is a human subject.
 75. The method of claim 64, wherein theisolated polypeptide is administered orally, intranasally, ocularly,nasally, transdermally, rectally, sublingually, or parentarally.